Tools for the diagnosis and treatment of Alzheimer&#39;s disease

ABSTRACT

The invention relates to epitopes of the tau protein which are specifically occurring in a phosphorylated state in tau protein from Alzheimer paired helical filaments, to protein kinases which are responsible for the phosphorylation of the amino acids of the tau protein giving rise to said epitopes, and to antibodies specific for said epitopes. The invention further relates to pharmaceutical compositions for the treatment or prevention of Alzheimer&#39;s disease, to diagnostic compositions and methods for the detection of Alzheimer&#39;s disease and to the use of said epitopes for the generation of antibodies specifically detecting Alzheimer tau protein. Additionally, the invention relates to methods for testing drugs effective in dissolving Alzheimer paired helical filaments or preventing the formation thereof.

This is a Divisional of U.S. application Ser. No. 08/244,603, filed Nov.28, 1994 now U.S. Pat. No. 6,200,788.

The invention relates to epitopes of the tau protein which arespecifically occurring in a phosphorylated state in tau protein fromAlzheimer paired helical filaments, to protein kinases which areresponsible for the phosphorylation of the amino acids of the tauprotein giving rise to said epitopes, and to antibodies specific forsaid epitopes. The invention further relates to pharmaceuticalcompositions for the treatment or prevention of Alzheimer's disease, todiagnostic compositions and methods for the detection of Alzheimer'sdisease and to the use of said epitopes for the generation of antibodiesspecifically detecting Alzheimer tau protein. Additionally, theinvention relates to methods for testing drugs effective in dissolvingAlzheimer paired helical filaments or preventing the formation thereof.

The brains of Alzheimer patients contain two characteristic types ofprotein deposits, the plaques and the tangles. These structures havebeen of peak importance in Alzheimer research during the last few years(for a recent review see Goedert et al., Current Opinion in Neurobiology1 (1991), 441 to 447). A prominent component of the tangles are thepaired helical filaments (PHFs). It seems now clear that the PHFs arelargely made up of the microtubule-associated protein tau which isnormally attached to the neuronal microtubule network and, furthermore,particularly enriched in the axons.

There are six isoforms of tau in human brain that arise from alternativesplicing of a single gene. All these isoforms also occur in PHFs(Goedert et al., Neuron 3 (1989), 519-526). The main biochemicaldifferences between normal and Alzheimer PHF tau protein known so farmay be summarized as follows:

-   (1) PHF tau protein is, in contrast to normal tau protein, highly    insoluble which makes a biochemical analysis difficult;-   (2) PHF tau protein reacts with certain antibodies in a    phosphorylation dependent manner, suggesting a special    phosphorylation status (Grundke-Iqbal et al., Proc. Natl. Acad. Sci.    USA 83 (1986), 4913-4917, Nukina et al., Proc. Natl. Acad. Sci. USA    84 (1987), 3415-3419);-   (3) PHF tau protein has a lower electrophoretic mobility in SDS    gels, suggesting a higher M_(r) value which may be related to its    phosphorylation pattern (Steiner et al., EMBO J. 9 (1990),    3539-3544);-   (4) PHF tau protein forms paired helical filaments with a    characteristic 78 nm crossover repeat (Crowther and Wischik, EMBO J.    4 (1985), 3661-3665).

Tau protein purified from brain has very little secondary structure (asjudged by CD spectroscopy), and a sedimentation constant of 2.6S,pointing to a highly asymmetric shape (Cleveland et al., J. Mol. Biol.1161 (1977), 227-247, in agreement with electron microscopic data(Hirokawa et al., J. Cell. Biol. 107 (1988), 1449-1459. The C-terminalhalf contains 3 or 4 internal repeats which are involved in microtubulebinding and promoting their assembly (hence “assembly domain”). Thisdomain can be phosphorylated by several protein kinases (Steiner et al.,EMBO J. 9 (1990), 3539-3544), a point that may be significant in view ofthe abnormal phosphorylation of Alzheimer tau (see, e.g. Grundke-Iqbalet al., ibid.). Moreover, the repeat region also lies in the core ofAlzheimer paired helical filaments (see, e.g. Goedert et al., ibid.;Jakes et al. EMBO J. 10 (1991), 2725-2729).

It has been hypothesized that PHF tau protein has a lower affinity formicrotubules compared to normal tau proteins since a similar effect hasbeen found when normal tau is phosphorylated in vitro by some kinases(Lindwall and Cole, J. Biol. Chem. 259 (1984), 5301-5305). Lack orreduced binding to microtubules might therefore be a result of abnormalphosphorylation of the tau protein. This abnormal state might lead tomicrotubule disassembly and interfere with vital neuronal processes,such as rapid axonal transport. The abnormally phosphorylated tauproteins might then aggregate into PHFs. As a consequence thereof theneurons would eventually die thus setting the stage for the generationof the Alzheimer's disease.

Up to now, it was not known which protein kinases are responsible forthe abnormal phosphorylation. Ishiguro et al. (Neuroscience Letters 128,(1991), 195-198) have isolated a kinase fraction from bovine brainextracts which contain a protein kinase recognizing the serine/threonineproline motif. This kinase phosphorylated residues Ser 144, Thr 147, Ser177 and Ser 315 of the tau protein. These residues differed from theones reported by others (Lee et al., Science 251 (1991), 675-678).Therefore, it remains unclear which protein kinase and which targetamino acid residue(s) are involved in the generation of Alzheimer'sdisease, if at all.

It is, moreover, of utmost importance for the diagnosis of Alzheimer'sdisease, in particular at an early stage of the disease process, todevelop antibodies which are specifically directed to epitopes on theprotein which are characteristic of the Alzheimer state. A monoclonalantibody, TAU1, has been isolated which is capable of distinguishingbetween phosphorylated and non-phosphorylated forms of the tau protein(see, e.g., Lee et al., ibid.). However, this antibody specificallyrecognizes dephosphorylated tau protein which is seemingly notassociated with the Alzheimer state. Another antibody, Alz 50(Ksiezak-Reding et al., J. Biol. Chem. 263 (1988), 7943-7947) reactswith PHFs as well as with tau protein. Sternberger et al., Proc. Natl.Acad. Sci. USA 82 (1985), 4774-4776, have isolated an antibody, SMI 34,which recognizes a phosphorylated epitope common to Alzheimer tauprotein and neurofilament protein. Finally, Lee et al. (ibid.) madeantibodies directed to a phosphorylated peptide comprising the KSPVmotif in the C-terminal region of the tau protein. All these antibodiesknown in the art have the disadvantage that for none of them it is knownwhether they recognize an epitope which is uniquely characteristic forthe Alzheimer's disease state.

Furthermore, no reliable data on the fine structure of Alzheimer pairedhelical filaments, nor on the mode or regulation of their formation fromtau proteins is available so far. For the prevention of the formation ofPHFs it would be highly advantageous if the mode of assembly of PHFsfrom tau protein and the regulatory mechanisms underlying said assemblywere known.

Thus, the technical problem underlying the present invention was toprovide a phosphorylated epitope characteristic for the Alzheimer tauprotein, a kinase activity which specifically catalyzes thisphosphorylation, pharmaceutical compositions comprising inhibitors tosaid kinases, antibodies for recognizing said epitopes, diagnosticcompositions containing said epitopes, methods involving kinases and/orantibodies for the in vitro diagnosis of Alzheimer's disease, methodsfor the in vitro conversion of normal tau protein into Alzheimer tauprotein and methods for testing drugs effective in dissolving AlzheimerPHFs or preventing the formation thereof.

The solution to the above technical problem is achieved by providing theembodiments characterized in the claims.) Accordingly, the presentinvention relates to an epitope of the tau protein which is specificallyoccurring in a phosphorylated state in tau protein from Alzheimer pairedhelical filaments.

The term “phosphorylated state in tau proteins from Alzheimer pairedhelical filaments” refers to a state of the tau protein where tau showsan upward M_(r) shift, has a reduced binding to microtubules and isphosphorylated at ser or thr followed by pro, or certain serines in therepeat region (see below).

Note: Amino acids are denoted by the one-letter or three-letter code;see e.g. Lehninger, Biochemistry, 2nd edition, Worth Publishers, NewYork, 1975, page 72.

There may be one or more epitopes of the tau protein which specificallyoccur in a phosphorylated state in Alzheimer paired helical filaments.These epitopes may, moreover, be phosphorylated by a single or differentenzymes displaying phosphorylating activity.

In a preferred embodiment of the present invention, said epitopes arespecifically phosphorylated by a protein kinase from mammalian brainhaving the following biochemical properties:

-   (a) it phosphorylates ser-pro and thr-pro motifs in tau protein;-   (b) it has an M_(r) of 42 kD;-   (c) it is activated by ATP and has a K_(m) of 1.5 mM;-   (d) it is activated by tyrosine phosphorylation;-   (e) it is recognized by an anti-MAP kinase antibody; and-   (f) it is deactivated by phosphatase PP2a.

The term “ser-pro and thr-pro motifs” as used herein refers to aphosphorylatable ser or thr residue followed by a pro residue. Thesetypes of sites are phosphorylated by the isoforms of MAP kinase, GSK-3,and cdk2 (see below).

The term “anti-MAP kinase antibody” refers to an antibody whichspecifically recognizes a mitogen activated protein kinase (MAP kinase).This kinase probably belongs to a family of closely related enzymeswhich have been referred to in the art by different names, e.g. MAP2(microtubule-associated protein 2, see e.g. de Miguel et al., DNA andCell Biology 10 (1991), 505-514) kinase, MBP (myelin basic protein)kinase or ERK1 (for a review, see Hunter, Meth. Enzym. 200 (1991),1-37). MAP kinase is similar with respect to its biochemical propertiesto functionally similar enzymes from a variety of sources (Hunter,ibid.).

In another preferred embodiment of the present invention said epitopeincludes the phosphorylatable serine residues 46, 199, 202, 235, 396,404 and/or 422 and/or the phosphorylatable threonine residues 50, 69,111, 153, 175, 181, 205, 212, 217 and/or 231; see FIG. 1 a.

The numbering of the amino acids was done in line with the largest humantau isoform, htau 40, see Goedert et al. (1989 ibid.).

In a particularly preferred embodiment said epitope includes thephosphorylatable serine residue of amino acid position 262. This isphosphorylated by the brain extract and the 35KD and 70KD kinasesprepared from it; see below. In accordance with the present invention ithas been shown that phosphorylation of said residue significantlyinterferes with binding of tau protein to microtubuli. This epitope maybe used for diagnostic in vitro methods to test for the onset ofAlzheimer disease.

In another particularly preferred embodiment said epitope includes thephosphorylatable serine residues 262, 293, 324 and 409.

Accordingly, another object of the invention is to provide a method fortesting the onset of Alzheimer disease by assaying the phosphorylationstatus of serine in position 262 and the other Ser-Pro or Thr-Pro motifsnamed above. This may e.g. be done by incubating a sample ofcerebrospinal fluid of a patient or a sample of nerve tissue afterbiopsy with a monoclonal or polyclonal antibody capable ofdistinguishing between a phosphorylated and a non-phosphorylated serine262 comprising epitope.

The epitopes of the invention may comprise one or more of the residuesenumerated above. Moreover, the epitopes of the present invention maycomprise only one or more phosphorylated serine residues, one or morephosphorylated threonine residues or a combination thereof. The actualcomposition of the epitope may be determined by methods which are knownin the art. It is also clear to the person skilled in the art that otheramino acids of the protein may contribute to the epitope which isrecognized by an antibody directed against the sites of tau proteinwhich are phosphorylated by MAP kinase.

In a further preferred embodiment of the present invention, said epitopecomprises the amino acid sequences

KESPLQ (SEQ ID NO: 2), YSSPGSP (SEQ ID NO: 3), PGSPGT (SEQ ID NO: 4),YSSPGSPGTPGS (SEQ ID NO: 5), PKSPSS (SEQ ID NO: 6), YKSPVVS (SEQ ID NO:7), GDTSPRH (SEQ ID NO: 8), MVDSPQL (SEQ ID NO: 9), PLQTPTE (SEQ ID NO:10), LKESPLQTPTED (SEQ ID NO: 11), AKSTPTA (SEQ ID NO: 12), IGDTPSL (SEQID NO: 13), KIATPRGA (SEQ ID NO: 14), PAKTPPA (SEQ ID NO: 15), APKTPPS(SEQ ID NO: 16), PAKTPPAPKTPPS (SEQ ID NO: 17), SPGTPGS (SEQ ID NO: 18),RSRTPSL (SEQ ID NO: 19), SLPTPPT (SEQ ID NO: 20), RSRTPSLPTPPT (SEQ IDNO: 21), VVRTPPK (SEQ ID NO: 22), VVRTPPKSPSSA (SEQ ID NO: 23),KIGSTENLK (SEQ ID NO: 24), KCGSKDNIK (SEQ ID NO: 25), KCGSLGNIH (SEQ IDNO: 26), KIGSLDNITH (SEQ ID NO: 27).

Again, it is to be understood that not all of the amino acids of thepeptide necessarily contribute to the specific site actually recognizedby the antibody.

Another object of the present invention is to provide a protein kinasewhich is capable of specifically converting tau protein to Alzheimer tauprotein by phosphorylation of the amino acid motif ser-pro or thr-pro.

Preferably, said protein kinase belongs to the class of MAP kinases.These kinases can be used for various purposes, e.g. for the in vitroconversion of tau protein into Alzheimer tau protein. The Alzheimer tauprotein thus obtainable may be used to study e.g. substances which arecapable of inhibiting its formation or the formation of PHFs. Moreover,they may be used for the development of drugs capable of dissolving saidPHFs or for converting Alzheimer tau protein into normal tau protein. Itis also conceivable that a system based on the ability of the proteinkinase of the invention to convert normal into Alzheimer tau proteinwill provide a well defined in vitro system for Alzheimer's disease.

In a preferred embodiment of the invention, said protein kinase has thefollowing biochemical properties:

-   (a) it phosphorylates ser-pro and thr-pro motifs in tau protein;-   (b) it has an M_(r) of 42 kD;-   (c) it is activated by ATP and has a K_(m) of 1.5 mM;-   (d) it is activated by tyrosine phosphorylation;-   (e) it is recognized by an anti-MAP kinase antibody; and-   (f) it is deactivated by phosphatase PP2a.-   The term “M_(r)” is defined as the relative molecular weight    determined by SDS gel.electrophoresis.

In still another preferred embodiment of the invention, said proteinkinase is obtainable by carrying out the following steps:

-   (a) homogenizing porcine brain in 10 mM Tris-HCl, pH 7,2, 5 mM EGTA,    2 mM DTT and a cocktail of protease inhibitors (leupeptin,    aprotinin, pepstatin A, α2-macroglobulin, PMSF (phenyl methyl    sulphonyl fluoride));-   (b) centrifugating the homogenate at 100,000×g for 30 minutes at 4°    C.;-   (c) removing the supernatant after centrifugation;-   (d) precipitating the crude protein by ammonium sulfate    precipitation;-   (e) desalting the crude preparation by gel filtration;-   (f) activating the crude enzyme by incubation in activation buffer;-   (g) further purifying the crude preparation by ion exchange    chromatography; and-   (h) identifying the enzyme by Western blotting.

The term “activation buffer” is defined as a buffer comprising 25 mMTris, 2 mM EGTA, 2 mM DDT, 40 mM p-nitro-phenylphosphate, 10 μM okadaicacid, 2 mM MgATP, and pro-tease inhibitors.

Another preferred embodiment of the present invention relates to aprotein kinase which is capable of specifically converting tau proteinto Alzheimer tau protein by phosphorylating IGS and/or CGS motifs in therepeat region of tau protein.

In a further preferred embodiment of the kinase of the invention, saidkinase is obtainable by carrying out the following steps:

-   (A) Subjecting mammalian brain extract to ion exchange    chromatography on Mono Q (Pharmacia);-   (B) testing the fractions eluted for binding to microtubules and    phosphorylation of the protein;-   (C) further purifying the fractions binding to microtubules and    capable of phosphorylating tau protein by gel chromatography;-   (D) subjecting the fraction eluting at about 35 kDal to ion exchange    chromatography on Mono Q;-   (E) collecting the major peak eluting between 200 and 250 mM NaCl;    and has the following characteristics:-   (a) it binds to Mono Q but not to Mono S;-   (b) it has an acidic pI;-   (c) it shows a major band (>95%) at 35 kDal and a minor band-   (<5%) at 41 kDal on silver-stained gels;-   (d) it incorporates a phosphate amount of 3.2 Pi into htau34, 3.4 Pi    into htau40, 3.3 Pi into htau23 and 2.8 Pi into mutant htau23    (Ser262→Ala); and    -   (e) it phosphorylates serine residues 262, 293, 324 and 409 of        tau protein.

Said brain extract may e.g. be human or bovine brain extract.

In still another preferred embodiment, the kinase of the presentinvention is obtainable by the following steps:

-   (A) preparation of high spin supernatant of extract from mammalian    brain;-   (B) subjecting the brain extract to chromatography on ion exchange    Q-Sepharose (Pharmacia);-   (C) testing the fractions and flowthrough for phosphorylation of tau    protein and influence on binding to microtubules;-   (D) chromatography of flowthrough on S-Sepharose, wherein the kinase    activity elutes at 250 mM NaCl;-   (E) chromatography on heparin agarose, wherein the kinase activity    elutes at 250 mM NaCl;-   (F) gel filtration, wherein the kinase activity elutes at 70 kDal;-   (G) chromatography on Mono Q, wherein the kinase activity elutes at    150 mM NaCl;    and has the following characteristics:-   (a) it does not bind to Q-Sepharose but to S-Sepharose;-   (b) it has an alkaline pI;-   (c) it shows a major band around 70 kDal on SDS gels;-   (d) it incorporates 3-4 phosphates into htau34, htau40, htau23, and    the construct K19 (i.e., the four-repeat microtubule binding    region);-   (e) it does not phosphorylate a mutant of K19 where Ser 262, 293,    324, and 409 are mutated into Ala; and-   (f) it phosphorylates Ser 262, 293, 324, and 409 or tau protein.

In another preferred embodiment of the invention, the 70 kDal kinasewhich phosphorylates the two IGS motifs and the two CGS motifs of tauprotein (Serines 262, 293, 324, 409) may be obtained as follows:

-   (A) Preparation of high spin supernatant of brain extract;-   (B) chromatography on Q-Sepharose;-   (C) chromatography of flowthrough on S-Sepharose, wherein the kinase    activity elutes at 250 mM NaCl;-   (D) chromatography on heparin agarose, wherein the kinase activity    elutes at 250 mM NaCl;-   (E) gel filtration, wherein the kinase activity elutes at 70 kDal;-   (F) chromatography on Mono Q, wherein the kinase activity elutes at    150 mM NaCl.    (See FIG. 45)

The brain extract in step A may be e.g. human or another mammalian brainextract.

The purification steps noted above are conventional ones known in theart as described throughout this specification.

Thus, preparation of the brain extract was carried out as described inExample 11, whereas binding studies between tau and taxol-stabilizedmicrotubules may be done as described in Example (6).

Furthermore, assays of tau-phosphorylation such as in-gel assays may becarried out as described in detail in Example 11.

Chromatography on Mono Q may be carried out as described in Example 11.

With respect to the actual conditions used for obtaining said kinase, aperson skilled in the art will be able to deviate from the protocoloutlined above and still obtain the kinase of the invention. Such adeviation may, e.g., concern the composition of the protease inhibitorcocktail of step (a): It is conceivable to use different inhibitorsunder the proviso that the kinase activity is not diminished ordestroyed.

In a most preferred embodiment the present invention relates to aprotein kinase which specifically phosphorylates serine residues 46,199, 202, 235, 262, 396, 404, 422 and threonine residues 50, 69, 111,153, 175, 181, 205, 212, 217, 231 of the tau protein.

In another most preferred embodiment, said kinase phosphorylates serineresidue 262.

A further preferred embodiment relates to a protein kinase which isglycogen synthase kinase-3, that is, isoform α, 51 kD or β (45 kD)and/or cdk2-cyclin A (33 kD).

In another preferred embodiment of the present invention, said kinase isa protein kinase from human brain, porcine brain, or another source.

Another object of the invention is to provide pharmaceuticalcompositions containing a specific inhibitor for the protein kinase ofthe invention, optionally in combination with a pharmaceuticallyacceptable carrier and/or diluent.

The term “specific inhibitor for the protein kinase” refers tosubstances which specifically inhibit the enzymatic action of theprotein kinase of the present invention. Inhibitors to enzymes such asprotein kinases and their mode of action are well known in the art. Forexample, such an inhibitor may bind to the catalytic domain of theenzyme thus rendering it incapable of converting its substrate. Examplesof such inhibitors are peptide inhibitors and deactivating phosphatasessuch as PP2a.

Another example is the deactivation of kinases by their phosphatases,e.g., PP-2a in the case of MAP kinase.

Said pharmaceutical composition may be administered to a patient in needthereof by a route and in a dosage which is deemed appropriate by thephysician familiar with the case. Pharmaceutically acceptable carriersand/or diluents are well known in the art and may be formulatedaccording to the route of administration or the special disease statusof the patient.

In a preferred embodiment the present invention relates to apharmaceutical composition for use in the treatment of Alzheimer'sdisease.

Again, said pharmaceutical composition may be administered to a patientin need thereof by route and in a dosage which is deemed appropriate bythe physician handling the case.

In another preferred embodiment of the present invention, saidpharmaceutical composition contains as the specific inhibitor at leastone oligo- or polypeptide comprising an epitope of the invention.

The term “oligo- or polypeptide comprising an epitope of the invention”refers to peptides which in their two- or three-dimensional structurereconstitute the epitope of the invention which is specificallyrecognized by an antibody directed thereto. Moreover, said oligo- orpolypeptides may solely consist of the amino acids representing saidepitope(s) or they may comprise additional amino acids. The constructionof such oligo- or polypeptides is well known in the art.

Another object of the invention is an antibody which specificallyrecognizes an epitope of the invention.

Said antibody may be a serum derived or a monoclonal anti-body. Theproduction of both monoclonal and polyclonal antibodies to a desiredepitope is well known in the art (see, e.g. Harlow and Lane, Antibodies,A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,1988). Furthermore, said antibody may be a natural or an antibodyderived by genetic engineering, such as a chimeric antibody derived bytechniques which are well understood in the art. Moreover, said antibodyalso refers to a fragment of an antibody which has retained its capacityto bind the specific epitope, such as a Fab fragment.

In a preferred embodiment, the antibody of the present inventionrecognizes the protein kinase of the present invention.

The term “recognizes the protein kinase of the present invention” asused herein means that the antibody does not or insignificantlycross-reacts with other substances such as different protein kinasespresent in the same biological environment. Moreover, it means that theantibody does not or insignificantly cross-reacts with different proteinkinases when tested in in vitro systems.

In another preferred embodiment, the antibody of the present inventionis a monoclonal antibody.

Another object of the invention is to provide diagnostic compositionsfor the detection and/or monitoring of Alzheimer's disease comprising

-   -   an epitope of the invention;    -   a kinase of the invention; and/or    -   an antibody of the invention.

The diagnostic composition of the invention may comprise for example anantibody of the invention which specifically recognizes one of thekinases of the invention or an enhanced level of said kinases in asample to be tested. In another embodiment, said diagnostic compositionmay comprise an antibody of the invention directed to one of theepitopes of the invention. Thus, an Alzheimer correlated disease stateof a sample may be detected by treating said sample with an antibodyrecognizing the epitope of the invention. The antibody-epitope (hapten)complex may be visualized using a second antibody directed to theantibody of the invention and being labelled according to methods knownin the art (see, e.g., Harlow and Lane, ibid.).

In still another embodiment of the present invention, said diagnosticcomposition may consist of an epitope of the invention and an antibodyof the invention. Treatment of a sample with said antibody may give riseto conclusions with regard to the disease state of the correspondingpatent, if the binding of said antibody to said sample is brought inrelation to binding of said antibody to said epitope of the inventionused as a reference sample.

In still another embodiment, the diagnostic composition may comprise anepitope of the invention, a kinase of the invention and an antibody ofthe invention. Kinase activity may be monitored with respect tophosphorylation of the sample as compared to the phosphorylation of theepitope of the invention. From the quantitated kinase activity thephosphorylation state of the tau protein contained in said sample andtherefore the disease state of the patient may be deduced. The kinaseactivity may e.g. be deduced by including a substrate analog in the samereaction, which is visually detectable upon enzymatic conversion. Suchsubstrate analogs are widely used in the art. Alternatively, the amountof phosphorylated tau protein in the sample may be detected aftertreatment with the kinase of the invention by employing an antibody ofthe invention directed to the phosphorylated epitope and using theamount of antibody-epitope complex provided by the diagnosticcomposition as an internal standard, or by determining the amount ofphosphate incorporated into tau protein by the kinase, e.g. byradio-active tracer methods which are well known in the art.

The person skilled in the art is in the position to design other testsystems which combine any of the above objects of the invention. It isto be understood that all conceivable combinations fall within the scopeof protection of the pre-sent invention.

Another object of the invention is to provide a method for the in vitrodiagnosis and/or monitoring of Alzheimer's disease comprising assaying acerebrospinal fluid isolate of a patient or carrying out a biopsy ofnerve tissue

-   -   for the presence of a phosphorylated Alzheimer tau protein        containing an epitope of the invention;    -   for the presence of a protein kinase of the invention; or    -   for the presence of phosphatases PP2a, PP1 and/or calcineurin.

The “cerebrospinal fluid isolate of a patient” is obtained by standardmedical procedures.

An example for a nerve tissue suitable for said biopsy is the olfactoryepithelium. The person skilled in the art may carry out said methodemploying e.g. the diagnostic tools illustrated in connection with thediagnostic compositions, supra.

In a preferred method of the present invention, the Alzheimer tauprotein and the phosphorylation of serine residue 262 of tau protein,respectively, is detected by using an antibody of the invention.

Said antibody preferably is an antibody directed to an epitope of theinvention.

In another preferred embodiment of the invention, the protein kinase isdetected by using an oligo- or polypeptide comprising an epitope of theinvention and/or by using an antibody of the invention.

Still another object of the invention is to provide a method for the invitro conversion of normal tau protein into Alzheimer tau proteinwherein normal tau protein is treated with a protein kinase of thepresent invention under conditions which allow the phosphorylation ofsaid normal tau protein.

The term “Alzheimer tau protein” refers to tau protein that isabnormally phosphorylated (e.g. at ser-pro or thr-pro motifs) andrecognized by Alzheimer-specific antibodies.

The term “conditions which allow the phosphorylation of said normal tauprotein” refers to conditions allowing the activity, preferably theoptimal activity, of protein kinase. This activity results inphosphorylation of the substrate at the ser-pro and/or thr-pro motifs.The phosphorylated substrate may then be recognized byAlzheimer-specific antibodies.

Normal tau protein may be derived from natural or recombinant sources.It is, for the purpose of carrying out the method of the presentinvention, however, expedient to use recombinant material.

The method of the present invention provides sufficient amounts ofAlzheimer tau protein for a variety of purposes: With the method of thepresent invention an in vitro model for the study of the generation ofthe Alzheimer state of proteins may be established (see above).Moreover, inhibitors may be tested which prevent the conversion ofnormal to Alzheimer tau protein. These “inhibitors” may be specific forthe epitope to be phosphorylated by e.g. blocking the epitope or may bedirected to various domains on the protein kinase, as long as theyprevent or disturb its biological activity. Another type of inhibitionis the antagonistic action of phosphatases on tau or its kinases.Furthermore, the Alzheimer tau protein generated by the method of thepresent invention may be employed in binding studies to microtubulestructures thus contributing to the elucidation of the molecular basisunderlying Alzheimer's disease.

The person skilled in the art knows how to employ the method of thepresent invention for a variety of different-purposes which all fallunder the scope of protection of the present invention.

The present invention relates, moreover, to the use of an epitope of theinvention for the generation of Alzheimer tau protein specificantibodies or antibodies to a tau protein specific for the onset ofAlzheimer disease.

The methods for obtaining said antibodies are well known in the art;thus, the generation of polyclonal or monoclonal antibodies may beconducted using standard methods (see, e.g., Harlow and Lane, ibid.). Ifan oligo- or polypeptide is used for the generation of antibodies it isdesirable to couple the peptide comprising the epitope to a suitablecarrier molecule capable of inducing or enhancing the immune response tosaid epitope, such as bovine serum albumin or keyhole limpet hemocyanin.The methods of coupling hapten (comprising or being identical to theepitope) and carrier are also well known in the art (Harlow and Lane,ibid.). It is also to be understood any animal suitable to generate thedesired antibodies may be used therefor.

In another aspect, the present invention relates to a pharmaceuticalcomposition for use in the treatment or prevention of Alzheimer'sdisease comprising an inhibitor of the formation of Alzheimer pairedhelical filaments from tau protein dimers.

In accordance with the present invention, it was found that tau proteinsform antiparallel dimers via assembly of their repeat units located inthe C-terminal domain of the protein. Whereas dimerization of tauproteins appears to be a physiological process, the formation of higherorder structures such as PHFs seems to be due to deregulation in theassembly process. Consequently, PHFs are formed from a number of taudimers wherein the cross-linking of dimers may occur via intermoleculardisulfide bridging.

Deregulation of the assembly process with subsequent formation of PHFsfrom tau dimers appears to be due to abnormal phosphorylation of tauproteins because, as has been found in accordance with the presentinvention, truncated tau proteins consisting merely of the repeat unitsare able to form PHFs, whereas tau proteins or tau-like proteinscomprising the N-terminus and C-terminus as well are unable to do so.

An inhibitor useful in the composition of the present invention istherefore any inhibitor capable of inhibiting the formation of PHFs fromtau dimers regardless of the molecular mechanism it interferes with.Such an inhibitor may be, for example, an inhibitor to a protein kinaseresponsible for abnormal phosphorylation of tau proteins as a compoundinterfering with the formation of intermolecular cross-links orassociation of tau dimers.

A further object of the present invention is to provide a method fortesting drugs effective in dissolving Alzheimer paired helical filamentscomprising the following steps:

-   (a) allowing the formation of Alzheimer paired helical filaments    from polypeptides comprising tau-derived sequences under appropriate    conditions;-   (b) incubating the Alzheimer paired helical filaments with the drug    to be tested; and-   (c) examining the result of the incubation of step (b) with respect    to the dissolution of the Alzheimer-like paired helical filaments.

The term “effective in dissolving Alzheimer paired helical filaments” asused herein is intended to also include partially dissolved PHFs. Forthe object of the present invention it is sufficient that the drug to betested is effective in the reduction of the size or the break-up ofPHFs, thus fulfilling a supplementary function in therapy, although atotal dissolution by the drug is preferred.

The term “polypeptides comprising tau derived sequences” refers to anypolypeptide which comprises sequences from tau protein capable offorming PHFs regardless of the length of said sequences or of mutations,deletions, insertions or heterologous sequences as long as the functionof said polypeptides to form PHFs remains intact.

The term “appropriate conditions” in connection with the formation ofAlzheimer PHFs refers to any condition which allows said formation. Saidconditions may include the availability of a MAP kinase if natural tauprotein is used.

In a preferred embodiment, the conditions applied in step (a) of saidmethod comprise an environment of 0.3 to 0.5 M Tris-HCl and pH 5.0 to5.5 without additional salts.

Still another object of the invention is to provide a method for testingdrugs effective in the prevention or reduction of the formation ofAlzheimer paired helical filaments comprising the following steps:

-   (a) incubating the drug to be tested with polypeptides comprising    tau-derived sequences under conditions which allow the formation of    Alzheimer paired helical filaments in the absence of said drug; and-   (b) examining the result of the incubation of step (a) with respect    to the presence or absence of Alzheimer paired helical filaments in    the incubation mixture.

The term “conditions which allow the formation of Alzheimer pairedhelical filaments in the absence of said drug” refers to any conditionwhich allows the formation of PHFs provided said drug is not included inthe incubation mixture. A preferred example of such a condition is anenvironment of 0.3 to 0.5 M Tris-HCl and pH 5.0 to 5.5 withoutadditional salts.

The term “presence or absence of Alzheimer paired helical filaments” asused herein is intended to include results wherein only a limited amountof PHFs has been formed as compared to control experiments where no suchdrug has been used.

In a preferred embodiment in the above methods, said polypeptidescomprise essentially the repeats from the C-terminal part of the tauprotein only.

In accordance with the present invention, it was found that the repeatscomprised in the C-terminal domain of the tau protein are responsiblefor dimerization of the protein under physiological conditions andsubsequent oligomerization leading to Alzheimer-like paired helicalfilaments. The term “Alzheimer-like paired helical filaments” is usedhere as opposed to “Alzheimer paired helical filament” solely toindicate that non-repeat unit parts of the tau protein normally presentin PHFs are absent from PHFs generated by said polypeptides.

Accordingly, the polypeptides comprising essentially the repeat unitsonly provide an ideal in vitro system to study PHF formation and studieson the fine structure of PHFs.

In a particularly preferred embodiment, said polypeptides are comprisingmainly the repeat regions of tau, such as K11 and/or K12.

K11 and K12 are ideally suited for the above testing purposes becausethey are essentially comprised of repeat units from the tau proteinonly.

For the method of the invention, K11 and K12 may be used alone or incombination.

In a further aspect, the present invention relates to a method fortesting drugs effective in dissolving Alzheimer paired helical filamentscomprising the following steps:

-   (a) introducing a functional gene encoding a MAP kinase under the    control of suitable regulatory regions into a cell expressing or    overexpressing tau protein;-   (b) allowing the formation of phosphorylated tau protein and of    Alzheimer paired helical filaments;-   (c) isolating said Alzheimer paired helical filaments;-   (d) applying the drug to be tested to said paired helical filaments    under appropriate conditions; and-   (e) examining the effect of said drug on said paired helical    filaments.

The term “cell expressing tau protein” as used in step (a), supra,refers to cells which endogenously express tau or which have thecapacity to express tau and into which a functional tau gene has beenintroduced. In the latter case the person skilled in the art is aware ofthe fact that the sequence of the introduction of the genes encoding theMAP-kinase and tau is irrelevant for the purpose of the method of theinvention.

The term “under appropriate conditions” in step (c), supra, refers toconditions which allow the drug to be effective in dissolving PHFs andare particularly optimal conditions.

Said method is particularly advantageous, since the system involvedwhich is based on the use of continuously growing cell lines providing aclose image of the in vitro situation provide an ample supply ofphosphorylated tau protein.

In a preferred embodiment said cell expressing tau protein is aneuroblastoma or chromocytoma cell or a primary culture of nerve cells.

Such cells or cell lines are well known in the art. Preferred examplesare the neuroblastoma cell lines N21 and PC12.

These cell lines are particularly preferred because they express tauendogenously.

A further object of the invention is a pharmaceutical composition forthe treatment of Alzheimer disease comprising a PP2a and/or PP-1 and/orcalcineurin phosphatase as the active or one of the active ingredients.

The Figures show:

FIG. 1A: Amino acid sequence of tau (SEQ ID NO: 1) (isoform htau40,Goedert et al., 1989). The motifs SP, TP, IGS and CGS are highlighted.

FIG. 1B: (A) SDS gel of tau isoforms, (B) immunoblots of (A) and PHF tauwith the AT8 antibody. (A) SDS gel. Lane 1, marker proteins. Lane 2: Taufrom bovine brain, showing several isoforms in a mixed state ofphosphorylation. Lane 3, bovine brain tau after dephosphorylation withalkaline phosphatase. Note that all isoforms shift to a lower M_(r).Lanes 4 and 5: Tau from normal human brain, before and afterdephosphorylation. Lanes 6-11: bacterially expressed human tau isoformshtau23, 24, 37, 34, 39, 40 (see Goedert et al., 1989, ibid.). Theseisoforms have either three or four internal repeats of 31 residues eachin the C-terminal half (three: htau23, 37, 39; four: htau24, 34, 40).Near the N-terminus there can be zero, one, or two inserts of 29residues (zero: htau23, 24; one: htau37, 34; two: htau39, 40).

(B) Immunoblots with the AT8 antibody. Lane 1, PHF tau, showing 4-6isoforms in the range of 60-70 kD; all of them react strongly with AT8.Lanes 2-11, same preparations as in (A); none of the bovine or normalhuman tau isoforms show any reaction.

FIG. 2: Phosphorylation of bacterially expressed human tau isoforms withthe kinase from brain. (A) SDS gels, (B) immunoblots with AT8.

(A) Lanes 1 and 2, SDS gel of htau23 before and after extractphosphorylation (note the upward shift in M_(r)) Lanes 3-10 showanalogous pairs for other isoforms (htau24, 34, 39, 40).

(B) Immunoblots of (A) with AT8 antibody. It reacts with all tauisoforms after phosphorylation (even lanes; including htau37, not shownhere).

FIG. 3: Diagram of constructs K3M, K10, K19, and K17. K19 (99 residues)contains the sequence Q244-E372 (SEQ ID NO: 28) of htau23 plus anN-terminal methionine. This comprises three of the repeats (repeat 1, 3,and 4; repeat 2 is absent in htau23). K10 (168 residues) is similar,except that it extends to the C-terminus of htau23 (L441). K17 (145residues) contains the sequence S198-E372 (assembly domain starting atthe chymotryptic cleavage site, up to end of fourth repeat, but withoutthe second repeat, plus an N-terminal methionine). K3M (335 residues)contains the N-terminal 154 residues of bovine tau4, plus the sequenceR221—L441 of htau23 (without second repeat). The location of peptideS198-T220 is indicated in K17. By comparison of the constructs theepitope of AT8 must be in this region (see FIG. 4).

FIG. 4: Phosphorylation of htau40 and constructs K10, K17, K3M, and K19.

(A) SDS gel. Odd lanes, htau40, K10, K17, and K3M beforephosphorylation, even lanes, after phosphorylation. Note the upwardshift of the bands after phosphorylation. In lane 4 there are two bandsbecause K10 is not completely phosphorylated.

(B) Immunoblot of (A) with AT8. The antibody reacts only with htau40(lane 2) and K17 (lane 6), both in the phosphorylated state, but notwith K10 (lane 4) or K3M (lane 8), although these constructs are alsophosphorylated and show an M_(r) shift.

(C) Construct K19 before and after incubation with the kinase. Lanes 1and 2, SDS gel; there is no M_(r) shift and no phosphorylation,confirmed by autoradiography (not shown). Lanes 3 and 4, immunoblot withAT8, showing no reaction. This confirms that the epitope is not in therepeat region.

FIG. 5: Diagram of tryptic peptide S195—R209. The 15 residue peptide(SEQ ID NO: 29) (containing 5 serines and 1 threonine) was labeled withtwo radioactive phosphates at S199 and S202, as determined bysequencing.

FIG. 6: Phosphorylation and antibody reactions of the D-mutant of htau23(S199 and S202 changed into D). Lanes 1 and 2, SDS gel of htau23 beforeand after extract phosphorylation; lanes 3 and 4, D-mutant before andafter extract phosphorylation. Note that the D-mutant runs slightlyhigher than htau23 (lanes 1,3), but after phosphorylation both proteinshave the same position in the gel (lanes 2, 4).

Lanes 5-8, immunoblots of lanes 1-4 with AT8. The antibody reacts onlywith extract phosphorylated htau23 (lane 6), but neither with theunphosphorylated form (lane 5) nor with the D-mutant (lanes 7, 8),although it was phosphorylated as seen by the additional shift andautoradiography (not shown).

Lanes 9-12, immunoblots of lanes 1-4 with TAU1. This antibody reactsonly with htau23 before phosphorylation (lane 9), but not with thephosphorylated form (lane 10) nor with the D-mutant (lanes 11, 12). Theaspartic acid apparently mimics a phosphorylated serine and thus masksthe epitope. The minor reaction of htau23 with TAU1 in lane 10 showsthat the protein is not completely phosphorylated.

FIG. 7: Time course of phosphorylation of bacterially expressed humanisoform htau23 with the brain kinase activity and correspondingautoradiogram.

(A) SDS-PAGE of htau23 after incubation with the kinase between 0 and 24hours, as indicated. The unphosphorylated protein is a single band ofM_(r)o=48 kD (lane 1). Lanes 3-14 show that phosphorylation leads to aprogressive shift to higher M_(r) with well defined intermediate stages.The even lanes (numbered 4, 6, etc. below FIG. 1B) are observed in thepresence of 10 μm okadaic acid (OA) (labeled “+” below FIG. 1A. The oddlanes (3, 5, etc. labeled “−”) are without okadaic acid. The first stagetakes about 2 hours (shift to a new M_(r)1=52 kD), the second isfinished around 10 hours (M_(r)2=54 kD), the third is finished aroundtime 24 hours (M_(r)3=56 kD); no further shift is observed during thesubsequent 24 hours. Lane 2 shows a mutant that is not of significancein this context.

(B) Autoradiogram of (A). The quantitation of the phosphate incorporated(mol P_(i)/mol protein) in this experiment was as follows (−OA/+OA): 30min (0.5/1.0), 60 min (0.7/1.4), 120 min (1.0/2.0), 10 hours (2.0/3.0),24 hours (3.2/4.0).

FIG. 8: (A) SDS gel showing the time course of phosphorylation of htau23similar to that of FIG. 1A, but with 10 μM okadaic acid throughout; (B)immunoblot of (A) with the monoclonal antibody SMI34. The antibodyrecognizes the protein only in the second and third stage ofphosphorylation, but not in the first.

FIG. 9: Binding of tau isoforms to microtubules before and afterphosphorylation.

(A) SDS gel of a binding experiment, illustrated for the case of the tauisoform htau40 (whose band is clearly separated from that of tubulin (T)so that both components can be shown simultaneously, without having toremove tubulin by a boiling step). The top line indicates pellets (P) orsupernatants (S), with or without phosphorylation for 24 hours (+ or−P_(i)). Lanes 1-4, 20 μM tau protein (total concentration),phosphorylated (lanes 1, 2) or not (lanes 3, 4). The comparison of lanes1 and 2 shows that most of the phosphorylated protein is free (S), whileonly a small fraction is bound to the microtubules (P). Lanes 3 and 4show that in the unphosphorylated state about half of the protein isbound, the other half free (note also that the phosphorylated proteinbands, lanes 1, 2, are higher in the gel than the unphosphorylated ones,lanes 3, 4, similar to FIG. 1). Lanes 5-8, similar experiment with 15 μMhtau40. Lanes 9, 10 show the case of 10 μM phosphorylated protein. Lanes11-15 are for density calibration with known amounts of htau40 (15, 10,7.5, 5, and 2.5 μM, resp.).

(B) Binding curves of htau23 and (C) htau34 to microtubules before(circles) and after 24 hour phosphorylation (triangles); these curveswere derived from SDS gels similar to that of FIG. 3A. Polymerizedtubulin is 30 μM. Fitted dissociation constants K_(d) andstoichiometries are as indicated. In each case the most dramatic effectis on the number of binding sites which decrease about three-fold uponphosphorylation, from around 0.5 (i.e. one tau for every two tubulindimers) down to about 0.16 (one tau for six tubulin dimers). Note thatthe binding of unphosphorylated 4-repeat isoforms (such as htau34) isparticularly tight (K_(d) round 1-2 μM).

FIG. 10: Diagram of htau40, showing the location of the 7 ser-pro motifsphosphorylated by the kinase activity. The boxes labeled 1-4 are theinternal repeats involved in microtubule binding; the second is absentin some isoforms (e.g. htau23). The two shaded boxes near the N-terminusare inserts absent in htau23 and htau24 so that these molecules haveonly 6 ser-pro motifs. The following radioactive tryptic peptides werefound:

24-49: KDQGGYTMHQOQEGOTDAGLKES.PLQ (SEQ ID NO: 31)

191-209: SGDRGYSS.PGS.PGTPGSR (SEQ ID NO: 32)

231-240: TPPKS.PSSAK (SEQ ID NO: 33)

396-405: SPVVSGDTS.PR (SEQ ID NO: 34)

385-405: TDHGAEIVYKS.PVVSGDTS.PR (SEQ ID NO: 35)

407-428: HLSNVSSTGSIDMVDS.PQLATL (SEQ ID NO: 36)

260-266: IGS.TENL (SEQ ID NO: 37)

FIG. 11: Binding of htau34 to microtubules, before (circles) and afterphosphorylation for 90 min (stage 1, triangles). The reduction inbinding capacity is very similar to that after 24 hours phosphorylation(compare FIG. 9B).

FIG. 12: SDS-PAGE and immunoblots of tau protein from Alzheimer andnormal human brain with antibodies SIM33, SMI31, and SMI34.

(A) Lane 1, SDS-PAGE of tau protein from a normal human control brain,showing 5-6 bands between M_(r)55 and 65 kD (somewhat lower than the PHFtau of lane 3). Lane 2, normal human tau after phosphorylation withkinase activity, resulting in an upward shift of all bands. Lanes 3, 4,immunoblot of PHF tau with antibody 5E2 which recognizes all tauisoforms independently of phosphorylation (Kosik et al., Neuron 1(1988), 817-825). Lane 3, PHF tau as isolated from an Alzheimer brain;lane 4, after dephosphorylation with alkaline phosphatase. Note that thebands of the dephosphorylated protein are shifted down on the gel.

(B) Immunoblot of (A) with SMI33. The antibody recognizes normal humantau (lane 1), and PHF tau after dephosphorylation (lane 4).

(C) Immunoblot of (A) with SMI31. Note that the antibody recognizesnormal human tau after phosphorylation, and PHF tau in its natural stateof phosphorylation (lanes 2, 3).

(D) Immunoblot of (A) with SMI34. This antibody recognizes normal humantau only after phosphorylation (lane 2), and PHF tau (lane 3).

FIG. 13: Time course of phosphorylation of bacterially expressed humanisoform htau23 (similar to previous figure) and immunoblots withantibodies SMI33, SMI31, SMI34, TAU1, and AT8.

(A) SDS-PAGE, phosphorylation times 0-24 hours, showing the successiveM_(r) shifts. (B-F) Immunoblots with SMI31, SMI34, SMI33, TAU1, and AT8.Antibodies SMI33 and TAU1 recognize htau23 fully up to the end of stage1 (2 hours), but the epitope becomes blocked during the second stage.Antibodies SMI31, SMI34, and AT8 are complementary in that theyrecognize the protein only in the second and third stage ofphosphorylation.

(G-H) Immunoblot of htau34 with SMI33 and SMI310 which recognize theprotein from the stage 2 phosphorylation onwards, similar to SMI31.

FIG. 14: SDS-PAGE of tau and several constructs, and immunoblots withthe antibodies SMI33, SMI31, and SMI34.

(A) SDS-PAGE. Lanes 1 and 2: Construct K10 before and afterphosphorylation with the kinase for 24 hours. Lanes 3 and 4: ConstructK17 before and after phosphorylation. Lanes 5 and 6: Construct K19before and after phosphorylation. All constructs except K19 show a shiftupon phosphorylation. With K10 one observes three shifted bands, withK17 there is only one shifted band.

(B) Immunoblot of (A) with SMI33: The antibody recognizes only K17 inthe unphosphorylated form (lane 3), suggesting that the epitope liesbefore the repeats.

(C) Immunoblot of (A) with SMI34. The antibody recognizes K10 and K17 inthe phosphorylated form (only top bands, lanes 2, 4). The antibody doesnot recognize K19 (the repeat region), but requires sequences on boththe N-terminal and C-terminal side of the repeats. The epitope istherefore non-contiguous (conformation-dependent).

(D) Immunoblot of (A) with SMI31. The antibody recognizes only the topband of the phosphorylated K10 (lane 2), suggesting that the epitopelies behind the repeat region.

FIG. 15: (A-G) Diagram of point mutants of htau40 and htau23.

FIG. 16: SDS gel of htau40 and the point mutants of FIG. 15, andimmunoblots with antibodies SMI33, SMI31, and SMI34.

(A) Lanes 1-8, SDS gel of htau40 and its mutants KAP235, KAP396, andKAP235/396 in the unphosphorylated and phosphorylated form (+). In eachcase phosphorylation leads to an upward shift in the SDS gel.

Blot of (A) with SMI33. The antibody response is strongly reduced whenS235 is mutated, both in the dephosphorylated and phosphorylated state(lanes 3+4, 7+8). This indicates that the (dephosphorylated) first KSPmotif is part of the epitope of SMI33. When S396 is mutated to A thebehavior is similar to the parent molecule, i.e. strong antibodyresponse in the dephosphorylated state, no reaction in thephosphorylated state, so that S396 does not contribute to the epitope ofSMI33.

(C Blot of (A) with SMI31. The antibody recognizes htau40 and allmutants in the phosphorylated form (lanes 2, 4, 6, 8). This shows thatphosphorylation of the two KSP motifs is not the main determinant of theepitope.

(D Blot of (A) with SMI34. The reaction is similar to SMI31 but morepronounced, again indicating that the two KSP motifs are not essential.

FIG. 17:: Deletion mutants of tau and their antibody response. (A) SDSgel of constructs containing only two repeats (K5-K7) or one repeat(K13-K15), before and after phosphorylation. (R) Immunoblot of (A) withSMI34. Note that the antibody recognizes all phosphorylated proteins (K7only weakly). (C) Immunoblot of (A) with SMI31. Note that the antibodyrecognizes the phosphorylated two-repeat molecules (K5-K5), but not theone-repeat molecules (K13-K15). Lanes 7 and 8 show htau40 as a control.(D) SDS gel of constructs K2, K3M, and K4, before and afterphosphorylation. (E) Blot of (D) with SMI34, recognizing only K4phosphorylated. (F) Blot of (D) with SMI31, recognizing only K2phosphorylated.

FIG. 18:(A-M) Diagram of htau40 and various mutants used in this study.

FIG. 19: Diagram of tau isoforms and constructs used in studies on taudimerization and oligomerization

(D) T8R-1,553 residues, MW 57743, derived from htau40 (see below). Ithas two inserts near the N-terminus (29 residues each, hatched), arepeat domain of four repeats (numbered 1-4) which is duplicated with asmall spacer in between.

(B) T8R-2,511 residues, MW 53459; it lacks the N-terminal inserts, buthas the four repeats duplicated.

(C) T7R-2,480 residues, MW 50212; similar to T8R-2, but without thesecond repeat sequence in the first repeat domain.

(D) Htau40, 441 residues, MW 45850, the largest of the six human tauisoforms (Goedert et al.), with two N-terminal inserts and a repeatdomain containing four repeats.

(E) Htau23,352 residues, MW 36760, the smallest of the human tauisoforms, without the N-terminal repeats and only three repeats.

(F) K11,152 residues, MW 16326, a repeat domain with four repeats plus ashort tail.

(G) K12,121 residues, MW 13079, a repeat domain with three repeats plusa short tail.

FIG. 20: SDS PAGE (4-20%) and gel chromatography of tau constructs andcross-linked products. Gels a and c were run in reducing conditions (3mM DTT in sample buffer), gel b in non-reducing conditions (except lane1 with 3 mM DTT in sample buffer).

(D) Constructs T8R-1, Htau23 and K12. Molecular weight markers are givenon the left.

(B) Construct K12 and cross-linked products. Cross-linking occursspontaneously in the absence of DTT; it can be prevented by DTT, orinduced by addition of PDM or MBS. Aggregation products are labeled onthe right (monomers, dimers, trimers, tetramers etc.).

(C) Silver stained SDS gel of a Superose 12 gel filtration run of K12cross-linked by PDM. The dimers (top band) elute before the monomers.Fractions 16 and 17 were used for electron microscopy.

(D) Elution profile of Superose 12 gel filtration of construct K12monomers and dimers cross-linked with PDM. The elution positions ofcalibration proteins are plotted against their effective hydrated Stokesradii on a logarithmic scale (right axis).

(E) CD spectrum of construct K12 (8 mg/ml in 40 mM HEPES pH 7.2, pathlength 0.01 mm). There is no significant α-helical or β-sheet structure.Similar spectra are obtained with other constructs as well as with fulllength tau.

FIG. 21: Synthetic paired helical filaments from construct K12.

(A) A tangle of synthetic PHFs from K12 (crossover period of.apprxeq.70-75 nm indicated by arrowheads). The construct was expressedand purified by the methods described previously (Steiner et al.). Itwas dialysed against 0.5 M Tris-HCl, with pH values between 5.0 and 5.5.The solution was negatively stained with 2% uranyl acetate.

(B) and (C) Single fibers of synthetic paired helical filaments madefrom construct K12. Note the crossover repeats (arrowheads) and therod-like particles of lengths around 100 nm (c, middle). Bar=100 nm.

FIG. 22 (A-C): Synthetic paired helical filaments from K12 dimerscross-linked with PDM and negatively stained with 1% phosphotungsticacid (micrographs provided by M. Kniel). Bar=100 nm.

FIG. 23: Paired helical filaments from Alzheimer brain (micrographsprovided by Dr. LichtenbergKraag).

(A) PHFs from neurofibrillary tangles prepared after Wischik et al.,stained with 1% phosphotungstic acid. This preparation containshomogeneous long filaments which still retain their pronase sensitive“fuzzy coat.” The crossover repeat is 75-80 nm, the width varies betweena minimum of about 10 nm and a maximum of 22 nm.

(B) PHFs prepared after Greenberg & Davies. This preparation results insoluble filaments of shorter length than in (A) and is moreheterogeneous. (1) is a paired helical filament with a 72 nm repeat anda width varying between 8 and 18 nm; (2) is a straight filament of 8 nmwidth; (3) is a twisted filament with a particularly wide diameter (upto 25 nm); (4) is a straight filament with a wide diameter (18 nm); (5)is a twisted rod-like particle about 80 nm long, equivalent to about onecrossover period. In many cases the particles appear to have brokenapart across the filament, e.g. the two rods labeled (4), the twistedfilament of (3) and the short stub to the right of it, or the twostraight rods above particle (3). Bar=100 nm.

FIG. 24: Electron micrographs of tau isoform htau23 and construct T8R-1prepared by glycerol spraying and metal shadowing

(A) monomers of htau23,

(B) dimers of htau23,

(C) monomers of T8R-1,

(D) folded forms of T8R-1 (hair-pin folds showing intramolecularantiparallel association),

(E) dimers of T8R-1. For lengths see Table 1 and FIG. 7. Interpretativediagrams are shown on the right. Bar=50 nm.

FIG. 25 (A-H): Length histograms of tau constructs and dimers.

FIG. 26: Electron micrographs of constructs K11 and K12.

(A) Monomers of K11,

(B) dimers of K11

(C) tetramers of K11 formed by longitudinal association of two dimers.

(C) Monomers of K12,

(D) dimers of K12,

(E) tetramers of K12. Bar=50 nm.

FIG. 27: (A) K12 dimers cross-linked by PDM (i.e. Cys322 to Cys322);

(B) K12 dimers cross-linked by MBS (i.e. Cys322 to nearby Lys). Bar=50nm.

FIG. 28: Antibody labeling of htau23, K12 and cross-linked productsthereof.

(A) htau23 dimers with an antibody at one end (left) and with anantibody at each end (right) demonstrating the antiparallel dimerizationof htau23;

(B) K12 diners with an antibody at one end (left), with antibodies atboth ends (middle) and presumable tetramers with antibodies at the freeends (right) indicating that this type of association blocks theepitope;

(C) K12 dimers cross-linked with PDM, with an antibody at one end(left), with antibodies at each end (middle) and a tetramer withantibodies at the free ends (right);

(D) K12 dimers cross-linked by MBS with an antibody at one end (left),with antibodies at each end (middle) and a tetramer with antibodies atthe free ends (right). Bar=50 nm.

FIG. 29 (A-G): Time course of phosphorylation of htau40 by GSK3 andimmune response. (A) SDS-PAGE of htau40 after incubation with the kinasebetween 0 and 20 hours at 37.degree. C. The minor lower band in lane 1is a fragment. Note the progressive shift to higher Mr values, similarto the effects of brain extract and MAP kinase. (B) Autoradiography. (C)Immunoblot with the antibody TAU1 whose reactivity is lost after.apprxeq.2 h (following the phosphorylation of S199 and S202). (D)Immunoblot with antibody AT-8. (E) Immunoblot with antibody SMI34(conformation sensitive and against phosphorylated Ser). (F) Blot withSMI31 (epitope includes phosphorylated S396 and S404). (G) Blot withantibody SMI33 which requires a dephosphorylated S235. There are somedifferences with respect to phosphorylation by MAP kinase or the brainextract. The SMI33 staining persists for a long period, suggesting thatSer235 is only slowly phosphorylated by GSK3. The staining of SMI31appears very quickly, before. that of ATS g4 or SMI34, showing that S396and S404 are among the earliest targets of GSK3.

FIG. 30 (A-B): Mobility shift of htau23 versus mutant htau23/A404 uponphosphorylation with GSK3. Top, SDS gel, bottom, autoradiogram. Lanes1-3, htau23 unphosphorylated and phosphorylated for 2 or 20 hours. Notethe pronounced shift and the clear incorporation of phosphate. Lanes4-6, mutant Ser404-Ala, unphosphorylated and phosphorylated for 2 and 20hours. The shift after 2 hours is much smaller and the degree ofphosphorylation much lower. This shows that the first strong shift andphosphorylation is at Ser404, similar as with MAP kinase and the brainextract kinase activity.

FIG. 31: Diagrams of tau constructs. Top, AP 17, a derivative of htau23with all Ser-Pro or Thr-Pro motifs altered into Ala-Pro. Middle, AP11,only Ser-Pro motifs changed into Ala-Pro. Bottom, K18, only 4 repeats oftau (derived from htau40).

FIG. 32: Copolymerization of MAP kinase and GSK3 with porcine brainmicrotubules. (A) SDS gel of microtubule purification stages. Ex=brainextract, supernatant after first cold spin. S=supernatant of first hotspin=tubulin and MAPs not assembled into microtubules after warming to37.degree. C.; P=pellet of redissolved microtubules. The other lanes (S,P) show two further cycles of assembly and disassembly by temperatureshifts (last pellet of microtubule protein was concentrated). (B) Blotwith anti-MAP kinase, showing mainly the p42 isoform and some of the p44isoform. (C) Blot with anti-GSK3B; note that this antibody shows somecross-reactivity with GSK3α. (D) Blot with anti-GSK3α. The blots showthat both kinases and their isoforms co-purify with the cycles ofmicrotubule assembly.

FIG. 33: (A) Identification of GSK3α and β in normal and Alzheimer brainextracts. M-markers, lane 1, SDS gel of normal brain extract, lane 2,immunoblot with anti-GSK3α; lane 3, immunoblot with anti-GSK3 β (withsome cross-reactivity to α). Lanes 4 and 5, same blots with Alzheimerbrain extracts. (B) Identification of GSK3 in association with PHF fromAlzheimer's tau.

FIG. 34: Binding curves of htau23 to microtubules (made from 10 μMtubulin in the presence of 20 μM taxol). Top curve (squares), htau 23unphosphorylated. Middle (circles), htau23 phosphorylated with GSK3,showing a comparable stoichiometry as the unmodified tau protein(saturating 0.6 per tubulin dimer). Bottom curve (triangles), control ofhtau23 phosphorylated with the brain kinase activity, showing apronounced decrease in stoichiometry. The solid lines show the best fitsassuming independent binding sites.

FIG. 35: (A) Diagram of htau23 and point mutants used in this invention.(B) Binding curves of htau23 and its point mutants to microtubules,unphosphorylated and phosphorylated with brain extract. The top andbottom curves show unphosphorylated and phosphorylated wild type htau23,the other curves are after phosphorylation. Mutants are (from top tobottom): Ser262-Ala, Ser235-Asp/Ser396-Asp, Ser404-Ala, Ser202-Ala. Themutation at Ser262 nearly eliminates the sensitivity of thetau-microtubule interaction to phosphorylation. These curves werederived from quantitating SDS gels by densitometry (see Example 6).Polymerized tubulin is 30 μM. The fitted stoichiometries n (=tau/tubulindimer) and binding constants K_(d)(μM) are:

htau23 wt non-phos. (n=0,49, K_(d)=2.5); A262 phos. (n=0.45, K_(d)=5.3);D235/D396 phos. A202 phos. (n=0.31, K_(d)=9.4); htau23 wt phos. (n=0.16,K_(d)=4.9).

FIG. 36: Binding curves of htau40 to microtubules. Top, unphosphorylatedhtau40 (triangles); middle, htau40 phosphorylated with MAP kinase(circles); bottom, htau40 phosphorylated with brain extract (squares).Fitted dissociation constants K_(d) and stoichiometries are asindicated.

FIG. 37: (A) Diagram of total mutant AP18. All Ser-Pro Thr-Pro arereplaced by Ala-Pro. In addition, Ser262 and 356 are mutated into Ala.In the mutant AP17 Ser262 and Ser356 remain unchanged. (B) Bindingcurves of htau 23 and the “total” mutants AP17 and AP18 to microtubuleswithout or with phosphorylation by brain extract. Top, unphosphorylatedhtau23 (filled triangles); middle, phosphorylated AP18 (circles), thetwo bottom curves are phosphorylated AP17 (open squares) and htau23(open triangles). The difference in behavior between AP17 and AP18 isdue to the phosphorylation of Ser262 in AP17. Fitted stoichiometries andbinding constants are:

htau23 wt non-phos. (n=0.49, K_(d)=2.5); AP18 phos (n=0.48, K_(d)=6.1);AP17 phos (n=0.18, K_(d)=6.6); htau23 wt phos. (n=0.16, K_(d)=4.9).

FIG. 38: Preparation of the kinase from porcine brain by chromatographicsteps. (A) Mono Q HR 10/10 FPLC. The phosphorylation of recombinanthuman tau 34 and construct AP17 is shown on the y-axis as moles P_(i)transferred per mole of tau. Fractions which decrease the binding of tauto MT elute around fraction 12, 20 and 30, the peaks around fractions 20and 30 being the most effective. (B) Fractions 28-32 from Mono Q weregel filtrated on a Superdex G-75 HiLoad 16/60 column. The column wascalibrated with standard proteins as shown by the filled symbols:Ribonuclease, 14 kDal; chymotrypsinogen A, 25 kDal; ovalbumin, 43 kDal;bovine serum albumin, 67 kDal. Molecular weight is indicate on the righty-axis on a logarithmic scale. The phosphorylation of htau34 andconstruct K18 is shown on the left y-axis. The highest activity elutesat a Mr of approx. 35 kDal. (C) Fractions 17-23 from the gel filtrationcolumn were pooled and rechromatographed on a Mono Q HR 5/5 column.Fraction 10 was used for binding studies. (D) SDS-gel showing the mainpurification stages. M: Marker proteins; lane 1, whole brain extract,lane 2, Mono Q HR 10/10 FPLC, fraction 30; lane 3, Superdex gelfiltration, fraction 22; lanes 4-5, Mono Q HR 5/5 FPLC, fractions 10 and9. Lane 5 shows the purified 35 kDal band and a trace at 41 kDal.

FIG. 39: SDS gel and in-gel assay of kinase activity (for details seeExample 11). (A) 7-15% silver stained SDS gel of fractions 9-11 (lanes1-3) of second Mono Q run (see FIG. 38 c). (B) Autoradiogram of anin-gel experiment, with tau construct K9 (=four repeats plus C-terminaltail of tau) in the gel and 51l each of fractions 9-11 (lanes 1-3). (C)Autoradiogram of control gel containing no tau protein and showing noautophosphorylation of the Mono Q fractions. Note that specific kinaseactivities are difficult to quantify from these gels since the renaturedprotein tends to diffuse out of the gels; this is especially true of the35 kDal band.

FIG. 40: Effect of phosphorylation of tau by 35 kDal kinase on gel shiftand microtubule binding. (A) SDS gel of htau23 and constructsphosphorylated by several kinases. M, marker proteins. Lanes 1 and 2,htau23 without and with phosphorylation by 35 kDal kinase. Lanes 3 and4, same experiment with point mutant htau23(Ser409-Ala) (no shift);lanes 5 and 6, point mutant htau23(Ser416-Ala) (only part of the proteinphosphorylated, but otherwise same shift as in lane 2); lanes 7 and 8,point mutant htau23(Ser404-Ala) (same shift as lanes 2 and 6). Themutants show that the 35 kDal kinase induces a shift by phosphorylatingSer409. Note that Ser404 is the target of MAP kinase, Ser416 of CaMkinase (Steiner et al., 1990, ibid.), and Ser409 and Ser416 of PKA, eachof which induces a shift. Lanes 9-11 show a comparison of the shiftsinduced in htau23 by the different kinases (CaM kinase, PKA and MAPkinase). The shifts induced by PKA (lane 10) is the same as that of the35 kDal kinase, and that MAP kinase produces by far the largest shift,typical of the Alzheimer-like state of tau. The bars on the rightindicate the shift level; from bottom to top, unphosphorylated htau23(control), CaM kinase shift level, PKA shift level, MAP kinase shiftlevel. All shift sites are near the C-terminus. (B) Binding curves ofhtau23 and the mutants Ser262-Ala to microtubules without or withphosphorylation by the kDal kinase (Mono Q fraction 10, 20 hours). Top,unphosphorylated htau23 (open circles, n=0.49, K_(d)=2,5 μM); middle,phosphorylated mutant (squares, n=0.44, K_(d)=11.6 μM); bottom,phosphorylated htau23 (filled circles, n=0.21, K_(d)=8.8 μM). In theabsence of Ser262 the reduction in stoichiometry is 0.05; withphosphorylated Ser262 it is 0.28.

FIG. 41: Diagram of htau40, highlighting the first microtubule-bindingrepeat (SEQ ID NO: 30) and the Ser262 that is important for microtubulebinding.

FIG. 42: 1. Dephosphorylation (“dephos.”) of p32-marked htau40(“ht40.sup.32P”) with different PPases. Autodiagraphs of 7-15% SDSgradient gels. FIG. 1: Autoradiographs of 7-15% SDS gradient gels.

A. Dephos. with PP2a H-isoform (10 μg/ml) Lane 1: ht40P before dephos.Lane 2: 10 min dephos. Lane 3: 30 min. dephos. Lane 4: 120 min dephos.

B. Dephos. with PP2a M-isoform (10 μg/ml), Lanes 1-4: see A.

C: Dephos. with PP2a L-isoform (10 μg/ml), Lanes 1-4: see A.

D: Dephos. with catalytic subunit of PP1 (500 U/ml), Lanes 1-4: see A.

FIG. 43: 2. Dephosphorylation with PP2a-H: disappearing ofphosphorylation dependent antibody epitopes

A. SDS-PAGE (7-15%). Lane 1: ht40P before dephos. Lane 2: 10 min dephos.Lane 3: 30 min. dephos. Lane 4: 120 min dephos. Lane 5: 5 h dephos. Lane6: 16 h dephos.

B. Autoradiographs

C. Immunoblot AT-8

D. Immunoblot Tau-1A

E. Immunoblot SMI-33

FIG. 44: Kinetics of dephos. with PP2a-H

A. time course of dephos. of ht40P with different concentrations of PP2a

B. variation in the ht40P-concentration: Michaelis-Menten-Diagramm.

FIG. 45: Preparation of the 70 kDal kinase which phosphorylates the twoIGS motifs and the two CGS motifs of tau protein (Serines 262, 293, 324,409). The kinase strongly reduces the affinity of tau for microtubules.

(A) Chromatography on S-Sepharose. Kinase activity elutes at 250 mMNaCl.

(B) Chromatography on heparin agarose. Kinase activity elutes at 250 mMNaCl.

(C) Gel filtration on Superdex G-75. Kinase activity elutes at 70 kDal.

FIG. 46: Time course of phosphorylation of htau40 with cdk2/cyclin A.Lanes 1-9 correspond to time points 0, 10, 30, 90 min, 3, 6, 10, 24hours, and 0 min (the 0 min lanes are the control).

(A) SDS polyacrylamide gel electrophoresis, showing the shift of theprotein upon phosphorylation.

(B) Autoradiogram showing increasing incorporation of phosphate.

(C) Immunoblot with TAU-1 antibody which recognizes onlyunphosphorylated Ser199 and Ser202.

(D) Immunoblot with AT-8 antibody which recognizes these two serines ina phosphorylated state, as well as Alzheimer tau.

EXAMPLE 1 Preparation of Tau Protein

Preparation of tau from normal brains: the Procedures of tau preparationfrom human, bovine, or porcine brain, dephosphorylation, andrephosphorylation were essentially as described by Hagestedt et al., J.Cell. Biol. 109 (1989), 1643-1651.

Preparation of tau from Alzheimer brains: Human Brain tissues fromneuropathologically confirmed cases of Alzheimer's disease were obtainedfrom various sources. The autopsies were performed between 1 and 25hours post mortem. The brain tissue was kept frozen at −70° C. Tau frompaired helical filaments (PHF) was prepared according to Greenberg &Davies, Proc. Natl. Acad. Sci. USA 87 (1990), 5827-5831.

EXAMPLE 2 Characterization and Partial Purification of the TauPhosphorylating Activity (Protein Kinase) of Porc Brain Extract

Porc brain extract supernatant was fractionated by ammonium sulphateprecipitation. The main kinase activity precipitated at 40% saturation.This fraction was desalted by gel filtration, diluted fivefold andincubated in activation buffer (25 mM Tris, 2 mM EGTA, 2 mM DTT, 40 mMp-nitrophenyl-phosphate, 10 μM okadaic acid, 2 mM MgATP, proteaseinhibitors) for 2 hours at 37° C. During this incubation aphosphorylation of a 44 kD protein at tyrosine residue(s) occurs asshown by Western blotting with anti-phosphotyrosine mAb. The 44 kDprotein could be identified as MAP2 kinase by a second mAb.

The crude enzyme activity was further purified by ion exchangechromatography (Mono Q FPLC, Pharmacia). Fractions containing theactivated MAP-Kinase, as shown by Western blotting, exerted the mostprominent tau phosphorylating activity (Peak I). A second tauphosphorylating activity (Peak II) did not induce comparable SDS-gelshifts and Alzheimer-specific antibody reactivity in tau.

EXAMPLE 3 Construction of Plasmids Carrying Genes Encoding RecombinantTau Polypeptides for the Determination of Alzheimer Tau Protein SpecificEpitopes

Cloning and expression of tau constructs: Plasmid preparations andcloning procedures were performed according to Sambrook et al.(Molecular Cloning Laboratory Handbook, 2nd edition, Cold Spring HarborLaboratory, Cold Spring Harbor, 1989). Amplifications by the polymerasechain reaction (PCR, Saiki et al., Science 239 (1988), 487-491) werecarried out using Taq polymerase as specified by the manufacturer(Perkin Elmer Cetus). The tau genes and their constructs were expressedin the expression vector pNG2, a derivative of pET-3b (Rosenberg et al.,Gene 56 (1987), 125-135), modified by removal of PstI, HindIII, NheI andEcoRV restriction sites for convenient engineering of the tau gene. Forthe expression the BL21 (DE3) E. coli system (Studier et al., Meth.Enzym. 185 (1990), 60-89) was used. Most constructs were derived fromthe human isoform htau23 which contains 352 residues and three internalrepeats in the C-terminal microtubule binding region (Goedert et al.,Proc. Natl., Acad. Sci. USA 85 (1988), 4051-4055). The numbering ofresidues used here refers to the sequence of htau40, the largest of thehuman isoforms (441 residues, Goedert et al., ibid.). For the isolationof the constructs use was made of the heat stability of the protein;they were separated by FPLC Mono S (Pharmacia) chromatography accordingto the procedure described by Hagestedt et al., J. Cell. Biol. 109(1989), 1643-1651.

K10: This represents the carboxy part of the htau23 isoform consistingof 168 residues (Q244-L441 plus start methionine, but without the secondrepeat V275-S305). The K10 tau cassette was generated in the pNG2/htau23vector by deletion of the NdeI-PstI fragment and replacing it with achemically synthesized hexamer 5′TATGCA3′. After religation the NdeIendonuclease site was restored and PstI site was damaged.

Constructs K11 and K12 were made by a combination of fragments derivedfrom the htau23 and htau24 genes. K11 is a tau derivative containing 4repeats and consists of 152 amino acids (Q244-Y394 plus startmethionine). K12 is a tau derivative containing 3 repeats and consistsof 121 amino acids (Q244-Y394 plus start methionine, but without thesecond repeat V275-S305, htau40 numbering).

Htau23 and htau40 are human tau isoforms consisting of 352 and 441 aminoacids, respectively (8).

K17: The K17 tau cassette (145 residues) is a shorter derivative of K16.It was made in two steps: First K16 was constructed using PCR toengineer the htau24 gene. The 5′ “add on” of restriction sites on bothends of the amplified fragment was applied to facilitate the insertionof the PCR products into the cloning vector. The start primer (JB50) hadthe sequence GGCG (“G/C clamp”), the CATATG recognition site for theNdeI nuclease (containing the universal ATG start codon), followed bycoding information for amino acids S198-T205. The stop primer (JB51) hada “G/C clamp” and the GGATCC recognition sequence for BamHI followed bya stop anticodon and anticoding sequence for the C terminal amino acidsP364-E372. The K16 tau cassette consists of 176 residues, 175 fromhtau40 (S198-E372) plus a start methionine. This fragment representspart of the assembly domain consisting of 46 residues between S198 andthe beginning of the first repeat following by the sequence of fourrepeats finished at E372. In the second step, a BstXI-BstXI fragmentfrom the newly constructed tau K16 cassette was exchanged against thesimilar BstXI-BstXI fragment from the htau23 gene containing only threerepeats and causing the generation of the tau cassette K17. Thus K17represents the analogous part of the projection domain like K16 butmissing the second tau repeat.

K3M (355 residues) is a chimera consisting of 145 residues from theamino terminus of bovine Tau4 (from the plasmid pETNde43-12, Himmler etal., Mol. Cell. Biol. 9 (1989), 1381-1388) and 190 residues from carboxypart of human htau23 (from the plasmid pUC18/htau23, Goedert et al.,1988 ibid.). It is a molecule with three repeats and two amino terminalinserts, consisting of 29 residues each. K3M was constructed by excisionof XmaI-BclI fragment from pETNde42-12 and replacing it with analogousXmaI-BclI fragment originated from the htau23 gene. This manipulationremoved 64 residues (XmaI-XmaI segment from bTau4) and replaced the 4repeats carboxy terminus against three repeats carboxy terminus.

K19 represents the three repeats of htau23 and consists of 99 residues(Q244-E372, plus start methionine, without repeat 2). The K19 moleculewas constructed from K17 by replacing the 144 nt long NdeI-PstI fragmentwith the synthetic hexamer 5′TATGCA3′. This modification retains theintact NdeI restriction site in the beginning of the molecule andremoves the PstI site.

Construction of the D-mutant of htau23: In order to replace S199 andS202 by D in htau23, a double stranded DNA cassette encoding the aminoacids G164-P219 was designed. This DNA segment was assembled from 8oligonucleotides (30 to 60 nucleotides in length) and contained SfiI andXmaI sticky ends. The insertion of the assembled cassette intolinearized pNG2/htau23 vector with removed native SfiI-XmaI fragmentcreated the required gene.

Construction of htau23/A404: htau23/A404 is a mutated htau23 moleculewhere Ser404 was replaced by the Ala in order to remove thisphosphorylation site. For convenient manipulation of the htau23 gene, anartificial NcoI restriction site in the position 1161 (htau40 numbering)was introduced. This mutation was done using PCR-SOE (splicing byoverlap extension, Higuchi et al., Nucl. Acids. Res. 16, (1988),7351-7367). The new NcoI does not influence the amino acid sequence oftau protein. For the introduction of the Ala residue in the position 404a synthetic DNA cassette was used, representing the 120 bp DNA fragmentbetween NcoI and NheI restriction sites and encoding the amino acidsHis388-Thr427. This DNA segment was assembled from 4 oligonucleotides(54 to 66 nucleotides in length) and contained NcoI and NheI stickyends. The insertion of the assembled cassette into the linearizedpNG2/htau23/NcoI vector with removed native NcoI-NheI fragment createdthe htau23/A404 gene. The mutation of Ser396 to Ala was created insimilar way like that in the position 404.

K2 (204 residues) is a chimera consisting of 36 residues from the aminoterminus of bovine Tau4 and 168 residues from the carboxy part ofhtau23; it contains three repeats. K4-K7 are deletion mutants of htau23containing only two repeats: K4 has repeats No. 1 and 3 (270 residues,D³⁴⁵-A426 excised); K5 has repeats No. 1 and 3 (310 residues, D345-T386excised); K6 has repeats No. 3 and 4 (322 residues, T245-K274 excised);K7 has repeats No. 1 and 4 (321 residues, V306-Q336 excised); note thatrepeat No. 2 is always absent in htau23. K13-K15 are deletion mutants ofhtau23 containing only one repeat: K13 has repeat No. 4 (291 residues,T245-Q336 excised); K14 has repeat No. 3(279 residues, T245-S305 andD345-D387 excised); K15 has repeat No. 1 (278 residues, D345-D387excised).

EXAMPLE 4 Determination of Alzheimer Specific Epitope in the Tau Protein

A panel of antibodies against PHFs from Alzheimer brain was closelyexamined for their reactivity and one (AT8) was found that was specificfor PHF tau. FIG. 1 shows the reactivity of the antibody AT8 againstdifferent tau species. In the case of tau from Alzheimer paired helicalfilaments (PHF) the antibody recognizes all isoforms (FIG. 1 b, lane 1).When the mixture of tau isoforms from normal bovine or human brain wastested (known to be in a mixed state of phosphorylation, FIG. 1 a, lanes2-5), reactivity with the AT8 antibody (FIG. 1 b) was detected. The sameis true for the six individual human isoforms expressed in E. coli(unphosphorylated, FIGS. 1 a and 1 b, lanes 6-11). It is concluded thatAT8 is indeed specific for Alzheimer tau; in particular, it reacts witha phosphorylated epitope that occurs only in PHFs, but not in normaltau. Moreover, there is a correlation between the AT8 reactivity,phosphorylation, and electrophoretic mobility; it appears as if therewas an Alzheimer-like phosphorylation that caused an upward shift in theSDS gel.

In order to identify the kinase(s) that were responsible for thisbehavior, and the corresponding phosphorylation sites, a kinase activityfrom porcine brain extract was prepared as described in Example 2. Thesix human isoforms expressed in E. coli were phosphorylated according tostandard procedures with this activity in the presence of okadaic acid,a phosphatase inhibitor. FIG. 2 a shows that each isoform changesconsiderably its electrophoretic mobility in the gel (upward shift) andshows a strong immunoreactivity with the AT8 antibody (FIG. 2 b). Theseresults show that the phosphorylation of tau by this kinase activity isanalogous to that of the Alzheimer state. Moreover, since all isoformsare affected in a similar way the phosphorylation site(s) must be in aregion common to all of them.

The strategy to identify said common region was to use first theengineered mutants prepared as described in Example 3 in order to narrowdown the site, and then to determine it by direct sequencing. FIG. 3describes some of the mutants used, K19, K10, K17, and K3M (see alsoExample 3). Except for K19, all of these mutants are phosphorylated bythe kinase activity and show an upward M shift in the SDS gel (FIG. 4a). K19 is a construct that comprises just three repeats of 31 or 32residues. It does not become phosphorylated by the kinase activity andtherefore does not show an M_(r) shift in the SDS gel (FIG. 4 c).

This means that the phosphorylation site(s) are outside the region ofthe repeats. Phosphorylation can take place on either side of therepeats and induces an upward shift in the gel; the shift is larger forphosphorylation after the repeats. The antibody AT8 recognizes none ofthe unphosphorylated forms (as expected); after phosphorylation itreacts only with the construct K17 (FIG. 4 b, lane 6), not with K10 orK3M (FIG. 4 b, lanes 4 and 8). In other words, K17 retains the epitope,while K10 and K3M have lost it. By reference to FIG. 3 it is concludedthat the epitope is not in the region of the pseudo-repeats nor inC-terminal tail where we found a CaM kinase site previously (since K10and K19 are non-reactive), but rather it has to be between S198 and T220(FIG. 3, peptide P), i.e. in the region following the major chymotrypticcleavage site (behind Y197) in the “assembly” domain of tau.

Next a total tryptic digest of radioactively labeled htau34, an isoformwith 4 internal repeats (Goedert et al., 1989, ibid.) was carried out.The peptides were isolated by HPLC and sequenced. One of them was in thearea of interest, S195—R209 (FIG. 5). This peptide contained twophosphates at S199 and S202. Both are followed by a proline, suggestingthat the enzyme active in the extract was a proline-directed kinase.

These results suggested that the phosphorylation sensitive AT8 epitopemight be in the vicinity of residue 200. This was tested by engineeringa mutant of htau22 (3 repeats, no N-terminal insert) where S199 and S202were both changed to D. This choice was made in order to rule out thephosphorylation of these residues by a kinase, but also to mimic in partthe “phosphorylated” state in terms of negative charges. On SDS gelsthis mutant showed a small upward shift to higher M (FIG. 6, lane 4).The immunoblots show that only the parent protein htau23 reacted withthe anti-body after phosphorylation (FIG. 6, lane 6), but not theunphosphorylated htau23 (as expected) nor the mutant, whetherphosphorylated or not (lanes 7, 8).

It is concluded that the epitope of AT8 is in the region S199-S202 anddepends on the phosphorylation of these two serines. They can bephosphorylated by a proline-directed kinase present in brain extractwhich turns the protein into an Alzheimer-like state. The region isperfectly conserved in all tau variants known so far and explains whyall of them respond to phosphorylation and to the antibody in the sameway.

EXAMPLE 5 Characterization of the Protein Kinase Activity

Phosphorylation of tau proteins was carried out in the following way:Tau protein (0.5 mg/ml) was incubated for various times (up to 24 hours)at 36° C. with the brain extract in 40 mM HEPES containing 2 mM MgCl₂, 1mM DTT, 5 mM EGTA, 1.5 mM PMSF, 2 mM ATP, 20 μg/ml protease inhibitormix (pepstatin, leupeptin, alpha-macroglobulin, aprotinin), with orwithout 1 mM okadaic acid. After that 500 mM DTT were added, thesolution was boiled for 10 min and centrifuged for 15 min at 15000 g at4° C. The supernatant was dialyzed against reassembly buffer (RB, 100 mMNa-PIPES pH 6.9, 1 mM EGTA, 1 mM GTP, 1 mM MgSO₄, 1 mM DTT) and used forbinding studies.

Radioactive labeling was done with gamma-[³²P]ATP (NEN Du Pont) at 10mCi/ml, 3000 Ci/mmol, diluted to 15-30 Ci/mol ATP for autoradiography onSDS gels. The phosphate incorporated into the protein was quantified asfollows: 1 μg of phosphorylated protein was applied to SDS gels, thebands were cut out and counted in the scintillation counter in Cerenkovmode. The counter was calibrated with known samples of ³²P (detectionefficiency about 50% in Cerenkov mode). The corrected counts weretranslated into moles of P_(i) per mole of tau on the basis of the knownspecific activity of radioactive ATP used during phosphorylation.

A remarkable feature found for this kinase is that it shifts the M_(r)of all tau isoforms in three distinct stages (see FIGS. 7 a and 8 a forthe case of htau23). During the first two hours of phosphorylation theprotein is converted from a M_(r0)=48 kD protein to a slower species,with an M_(r1) of about 52 kD. Upon completion of this first stage, asecond one sets in which is finished around 6.10 hours (M_(r2)=54 kD),The third stage takes about 24 hours (M_(r3)=56 kD), after that no moreshift is observed.

During the initial stage each band of the tau doublet incorporatesphosphate (e.g. at a level of about 0.5 P_(i) per molecule in thepresence of OA at 30 min, see FIG. 7 b, lane 4). This means that theremust be at least two distinct phosphorylation sites, one that isresponsible for the shift (the “shift site”, upper band), and one thathas no effect on the M_(r) (lower band). The lower band graduallydisappears, and at two hours each tau molecule contains about 2 P_(i).In other words, the upper band contains tau molecules in which the“shift site” is phosphorylated, irrespective of the other site(s);whereas the lower band contains only molecules where the shift site isnot phosphorylated. The effect of OA is seen mainly in the lower band,indicating that the phosphatase operates mainly on the non-shiftsite(s). These considerations apply to the first stage ofphosphorylation; during the second and third stages there are furthershifts, but a detailed analysis of shift sites and non-shift sites isnot possible because of the overlap of bands. Overall about twoadditional phosphates can be incorporated in every stage, giving amaximum of 6 for htau23 and 7 for htau34. These values refers to thepresence of OA; without it we usually find ≈1-2 P_(i) less. When thepurified kinase is used, one finds 12-14 P_(i).

Since the major shift occurs during the first stage, and since a largeshift is considered a hallmark of Alzheimer tau, it was suspected thatthe first stage phosphorylation might induce an Alzheimer-like state.This was checked by immunoblotting according to standard procedures withAlzheimer-specific antibodies. FIG. 8 a shows a similar phosphorylationexperiment as above (with 10 μm OA throughout), FIG. 8 b is theimmunoblot with the monoclonal antibody SMI34 which reacts with aphosphorylated epitope in Alzheimer tangles (Sternberger et al., ibid.).The antibody recognizes the bacterially expressed tau phosphorylated bythe kinase, but only from stage 2 onwards. A similar behavior is foundwith other Alzheimer-specific antibodies tested. The result from thesestudies is that the major phosphorylation-dependent M_(r) shift(stage 1) is distinct from the ones that generate the Alzheimer-likeantibody response (stages 2, 3).

EXAMPLE 6 Tau Protein in Microtubule Binding Studies

Another point of interest with respect to the correlation betweenabnormal phosphorylation of tau proteins and Alzheimer's disease waswhether the phosphorylation had an influence on tau's affinity formicrotubules. This was tested using a microtubule binding assay.Accordingly, PC tubulin was incubated at 37° C. in the presence of 1 mMGTP and 20 μM taxol. After 10 min tau protein was added in differentconcentrations and incubated for another 10 min. The suspensions werecentrifuged for 35 min at 43000 g at 37° C. The resulting pellets wereresuspended in CB buffer (50 mM PIPES PH 6.9, 1 mM EGTA, 0.2 mM MgCl₂, 5mM DTT, 500 mM NaCl). In the case of htau 23 and htau 34 the pellets andsupernatants were boiled for 10 min and recentrifuged for 10 min at43000 g at 4° C. (this step served to remove the tubulin component whichotherwise would overlap with these tau isoforms on SDS gels). Pelletsand supernatants (containing the bound and the free tau, respectively)were subjected to SDS PAGE (gradient 7-15% acrylamide) and stained withCoomassie brilliant blue R250. The gels were scanned at 400 dpi on anEpson GT 6000 scanner and evaluated on a PC 368AT using the programGelScan (G. Spieker, Aachen). The protein concentration on the gel wasalways within the linear range (up to 1.5 optical density). Theintensities were transformed to concentrations using calibration curvesand used in the binding equation.

Tau_(bound)=n[Mt][Tau_(free)]/{Kd+[Tau_(free)]}, from which thedissociation constand K_(d) and the number n of binding sites per dimerwere obtained by fitting. [Mt] is the concentration of tubulin dimerspolymerized in microtubules (usually 30 μM).

With fully phosphorylated protein (stage 3, 24 hours) a dramaticdecrease in binding capacity of htau23 was observed (FIG. 9 b), fromabout one tau per two tubulin dimers to one tau per six tubulin dimers.In other words, it appears that unphosphorylated tau packs tightly ontoa microtubule surface, whereas fully phosphorylated tau covers themicrotubule surface less densely, as if it occupied more space. FIG. 9 cshows the same experiment with htau34. The results are similar, i.e.there is a threefold reduction in binding capacity. Tau isoforms withfour repeats, such as htau34, bind to microtubules particularly tightlyin the unphosphorylated state (K_(d) ⁻1-2 μM).

Since the major M_(r) shift (see Example 5) occurs during the initialtwo hours it was of interest to find out which residues becomephosphorylated during the first stage, and how they affected microtubulebinding. As mentioned above, there are about two phospates incorporatedduring this period, one of which causes the shift from M_(r0) to M_(r1).FIG. 10 illustrates the binding of htau34 to microtubules after 90 minof phosphorylation. The striking result is that the limitedphosphorylation decreases the affinity as efficiently as the fullphosphorylation. This means that the reduction in microtubule affinityprecedes the Alzheimer like immunoreactivity (FIG. 8).

The analysis of tryptic peptides after 90 min showed four major peaks ofradioactivity, with phosphates on serines 202, 235, 404, and 262. Threeof these are SP sites that are not in the repeat region, but ratherflank that region in nearly symmetric positions (FIG. 11); the fourth(S262) is a non-SP site in the first repeat. It is in particularnote-worthy that S396 was not among the phosphorylated residues. Thiswas unexpected since Lee et al. (1991, ibid.) had shown that S396 (thecenter of a KSP motif) was phosphorylated in tau from paired helicalfilaments. Thus S396 must become phosphorylated during the second orthird stages of phosphorylation, concomitant with the immunoreactivity(FIG. 8 b).

Several point mutants were generated according to standard procedures tofind out which site(s) were responsible for the initial M_(r) shift.When ser404 was turned into ala the M_(r) shift during the first stagedisappeared, whereas it remained visible when ser199, 202, 235, or 396were mutated. This means that the phosphorylation of ser404 accounts forthe one P_(i) present in the upper band of FIG. 7 a or 8 a. Theadditional ≈1P_(i) present after 2 hours is distributed among serines202, 235, and 404.

Whereas the results on the “shift site” S404 of tau are clear cut, thefactors responsible for the reduction of microtubule binding are morecomplex. The S404-A mutant binds to microtubules similarly as the parenthtau34; after 90 min of phosphorylation the stoichiometry decreasesabout 2-fold, i.e. less than the factor of 3 observed with the parentmolecule. If S404 were the only residue whose phosphorylation wasresponsible for the loss of microtubule binding we would not expect anydecrease in the mutant. The fact that a decrease is observed means thatother factors play a role as well; these factors are presumably relatedto the incorporation of more than one P_(i) at one or more of the othersites before or at the beginning of the repeat region (e.g. 202, 235,262). However, these residues cannot by themselves be responsible forthe full decrease of affinity either. In fact, point mutations atpositions 202 or 235 show a similar effect as that of 404, i.e. only apartial reduction of binding. One possible explanation is that differentphosphorylation sites interact in a cooperative manner and generate anew confirmation.

EXAMPLE 7 Time Course of Phosphorylation as Determined by Stage SpecificAntibodies

Neurofilament specific antibodies SMI31, SMI34, SMI35 and SMI310 againsta phosphorylated epitope and SMI33 against a non-phosphorylated epitope[(Sternberger et al., Proc. Natl. Acad. Sci. USA 82 (1985), 4274-4276)]were used to detect stage specific phosphorylation of tau protein. SMI33recognizes normal human brain tau (FIG. 12, lane 1) but does notrecognize PHF tau, except when it is dephosphorylated (lane 4). Thissuggests that the epitope of SMI33 is specifically blocked by somephosphorylation in the Alzheimer state which does not occur in normalbrain tau. SMI31 and SMI34 both react in a complementary fashion toSMI33: Only PHF tau is recognized (FIGS. 12 c and 12 d, lane 3), but notwhen it is dephosphorylated (lane 4), nor the normal tau control (lane1).

The testing of the various antibodies during the time course ofphosphorylation shows that SMI33 loses reactivity during the secondstage of phosphorylation (see FIG. 7).

For antibody SMI31 no reactivity is observed with the unphosphorylatedprotein (time 0) or during the first stage, but the reactivity appearsgradually during the second stage and remains throughout the third. Asimilar time course is found with antibody SMI34 (FIG. 13 c and compareFIG. 12 d, lane 3), SMI35, and SMI310 (FIGS. 13 g,h). For comparison theblots with AT8 (FIG. 13 f), a phosphorylation sensitive Alzheimer tangleantibody (Binder et al., J. Cell. Biol. 101 (1985), 1371-1379 areincluded) and TAU1, an antibody against dephosphorylated tau. AT8 reactssimilarly to SMI31, SMI34, SMI35, and SMI310, while TAU1 is similar toSMI33. The striking feature of the blots is that in each case it is thestage 2 phosphorylation that determines the antibody response.

These experiments could be interpreted by assuming that the antibodiesreact with the same region of tau in a dephosphorylated orphosphorylated form; but this assumption is too simple, as shown later.Two other features should be pointed out, however: One is that thelargest gel shift (stage 1) is not the one that causes theAlzheimer-like immunoreactivity (appearing in stage 2). Thus not everygel shift of tau is diagnostic of the Alzheimer state, althoughconversely the Alzheimer state always shows a gel shift. Secondly, thereis a surprisingly precise relationship between gel shift,phosphorylation, and immunoreactivity with several different antibodies.

The major phosphorylated motifs of neurofilaments are repeated sequencesof the type KSPV (SEQ ID NO. 38) where S is the phosphate acceptor; seee.g. Geisler et al., FEBS Lett. 221 (1987), 403-407. Tau has one suchmotif, centered at S396, and another KSP motif is centered at S235. Thetwo KSP sites lie on either side of the repeat region and are conservedin all tau isoforms. By analogy one may suspect that these sites areinvolved in the reaction with the SMI antibodies that were raisedagainst neurofilaments. We tested this in three ways, by mutating one ortwo of the serines, by making smaller tau constructs, and by directsequencing of tryptic peptides.

Constructs K10, K17, and K19 were examined before or afterphosphorylation with the kinase (FIG. 14 a). K10 and K17 show an M_(r)shift, but not K19. Note also that K10 and K17 are only partly convertedto the higher M_(r) form in this experiment, indicating that theirphosphorylation is less efficient. K10 shows three shifted bands,indicating that there are three phosphorylation sites in the C-terminalregion. K17 shows only one shifted band so that there is only oneshift-inducing site in the region before the repeats. FIGS. 14 b-d showthe immunoblots with SMI33, SMI31, and SMI34; the data on SMI35 andSMI310 are similar to SMI31 (not shown). Antibody SMI33 reacts only withK17 in the dephosphorylated state, but not with K10 and K19 (FIG. 14,lane 3). This suggests that the epitope is in a region before therepeats, between S198 and Q244, outside the sequences covered by theother constructs. This would be consistent with an epitope at the firstKSP site. Antibody SMI31 reacts with K10 in its phosphorylated form, butnot K17 or K19 (FIG. 14). Using similar arguments as before, the epitopeis in the region T373-L441, consistent with the second KSP site.Finally, antibody SMI34 labels htau23, K10 and K17, but not K19 (FIG. 14c). The latter property would argue against the repeat region as anepitope, but the remaining reaction with K10 and K17 would seem mutuallyexclusive. Our interpretations is that SMI34 has a conformationalepitope that depends on tails on either side of the repeats and becomesfully stabilized only when at least one tail is present. However, thephosphorylation dependence is in each case the same as that of theintact molecule.

Since it was suspected that the two KSP motifs were phosphorylated bythe kinase, it was tried to prove this directly. Radioactively labeledtryptic peptides of htau34 were identified by HPLC and proteinsequencing, and phosphorylated residues were determined. There are twomajor phosphorylated tryptic peptides in these regions; peptide 1(T231-K240, TPPKS _(p) PSSAK (SEQ ID NO.: 33)) contains the first KSPmotif, phosphorylated at S235, peptide 2 (T386-R406, TDHGAEIVYKS _(p)PVVSGDTS _(p) PR (SEQ ID NO: 35)) contains the second KSP site,phosphorylated at S396 and S404. S416, the single phosphorylation siteof CaM kinase described earlier (Steiner et al., EMBO J. 9 (1990),3539-3544, S405 in the numbering of htau23 used earlier) is notphosphorylated by the kinase used here.

Next point mutants of the phosphorylated residues 235 and 396 (FIG. 15)were made and analysed in terms of gel shift and antibody reactivity(FIG. 16). The parent protein htau40 and its KAP mutants have nearlyidentical M_(r) values, and they all shift by the same amount afterphosphorylation (FIG. 16, lanes 1-8). The reactivity of SMI33 isstrongly reduced when S235 is mutated to A (FIG. 16, lanes 3, 7) andobliterated after phosphorylation (FIG. 16, lanes 2, 4, 6, 8). Thismeans that the epitope of SMI33 is around the first KSP site, butphosphorylation at other sites have an influence as well (perhaps via aconformation). The mutation at S396 (second KSP site) has no noticeableinfluence on the SMI33 staining (FIG. 16 b, lanes 5, 6).

As mentioned above, the epitope of SMI31 depends on the phosphorylationof sites behind the repeat region. When S396 is mutated to Ala theantibody still reacts in phosphorylation dependent manner so that thisserine is not responsible for the epitope by itself (FIG. 16 c, lane 6).Mutation S404 to Ala yields the same result. However, if both serinesare mutated, the antibody no longer reacts upon phosphorylation (notshown). This means that the epitope includes the two phosphorylatedserines. The binding of this antibody also has a conformationalcomponent: constructs with only one repeat (K13-K15) are not recognized(FIG. 17, lanes 10, 12, 14).

SMI34 shows the most complex behavior because its reactivity depends onphosphorylation sites before and after the repeat region. This antibodyrecognizes all KAP mutats, so that S235 and S396 cannot play a majorrole. However, the fact that SMI34 recognizes phosphorylated K17, K10,but not K19 (FIG. 17) suggests that the regions before and/or behind therepeats must cooperate with the repeats to generate the epitope. Onepossibility would be that the epitope is non-contiguous, another one isthat it may depend on the number and conformation of the repeats. Inorder to check these possibilities constructs with differentcombinations of two repeats (K5, K6, K7, FIG. 18), and constructs withone repeat only (K13, K14, K15) were done. All of these showed a shiftupon phosphorylation, and all of them were recognized by SMI34 (thereaction is less pronounced when the third repeat is absent, indicatingthat this repeat is particularly important for the conformation, FIG.17, lane 6). This means that the epitope of SMI34 does not depend on thenumber of repeats. However, the nature of the region just before therepeats seems to be important and in particular sensitive to charges.This can be deduced from constructs such as K2 or K3M where chargedsequences have been brought close to the repeat region, resulting in aloss of SMI34 reactivity. In other words, it seems as if the chargedsequences are capable of masking the epitope, independently of thephosphorylation itself (FIG. 17, lanes 2, 4). The interactions betweenthe constructs and the antibodies are summarized in Table 1.

EXAMPLE 8

Cloning and expression of tau constructs: Plasmid preparations andcloning procedures were performed according to Sambrook et al. PCRamplifications were carried out using Taq polymerase as specified by themanufacturer (Perkin Elmer Cetus) and a DNA TRIO-Thermoblock (Biometra).

Tau cDNA clones and their constructs were subcloned into the expressionvector pNG2 (a derivative of pET-3b, Studier et al., modified in thelaboratory by removal of PstI, HindIII, NheI and EcoRV restriction sitesfor convenient engineering of the tau clones), or in expression vectorpET-3a. For the expression, the BL21 (DE3) E. coli system was used(Studier et al.). All residue numbers refer to the sequence of htau40,the largest of the human isoforms (441 residues, Goedert et al.). Forthe isolation of the constructs the heat stability of the protein wasused; they were separated by FPLC Mono S (Pharmacia) chromatography (fordetails see Hagestedt et al.).

Construction of T8R-1: This is a tau derivative containing 8 repeats. Itwas constructed on the basis of the bovine tau4 isoform (Himmler etal.). Two plasmids, pETNde43-12 (containing the btau4 gene) and pET-KO(containing KO which consists mainly of the four repeats plus leadingand trailing sequences from the vector, Steiner et al.) were used forthe construction of T8R-1. The NdeI-RsaI DNA fragment from btau4 wasconnected with “filled in” XmaI-BamHI fragment of KO leading to chimericmolecule consisting of 553 amino acids. The T8R-1 gene encodesMet1-Bal393 connected through the artificially introduced Ser residuewith the Gly248-Tyr394 tau fragment, followed by a 23 residue tail fromthe bacteriophage T7 sequence (htau40 numbering).

Construction of T7R-2 and T8R-2: T7R-2 is a tau derivative containing 7repeats, T8R-2 contains 8 repeats. Both molecules were constructed onthe basis of the human htau23 and htau24 isoforms (Goedert et al.). Forthe engineering of the T7R-2 and T8R-2 molecules, PCR repeat cassettesA1 (encoding 4 repeats), A2 (encoding the whole carboxy part of thetau24 molecule including the four repeat sequence and the tau sequencebehind them) and A3 (encoding 3 repeats) were prepared. The T8R-2molecule was generated by combination of A1 and A2 with NdeI-PstI DNAfragment isolated from htau23. This tau derivative consists of 511 aminoacids, the first 311 N-terminal residues of htau24 (Met1-Lys369,containing 4 repeats), followed by Gly-Thr link, then by 198 residues ofthe C-terminus of htau24 (Gln244-Leu441, four more repeats). The T7R-2gene was generated similarly to T8R-2 but the A3 cassette was usedinstead of A1. The T7R-2 protein consists of 480 amino acids, the first280 N-terminal residues of htau23 (Met1-Lys369, including 3 repeats),followed by Gly-Thr link, then by 198 residues of the carboxy terminalpart of htau 24 (gln244-Leu441, containing 4 repeats, htau40 numbering).

EXAMPLE 9 Conformation of Tau Protein and Higher Order StructuresThereof

(a) Conformation and Dimerization of Tau Constructs

FIG. 19 illustrates the types of constructs used in this example. Threetypes of molecules were used: (i) tau isoforms occurring in brainranging from htau23 (the smallest isoform, 352 residues) to htau40 (thelargest 441 residue, see Goedert et al.). They differ mainly by thenumber of internal repeats in the C-terminal domain (3 or 4) and thenumber of inserts near the N-terminus (0, 1, or 2). The internal repeatsare involved in microtubule binding and in the formation of pairedhelical filaments; attention was then focused on the those constructswhich would presumably yield information on the structure of the repeatregion; (ii) engineered constructs with an increased number of repeats,e.g. seven or eight (T7, T8); (iii) constructs containing essentiallythe repeats only (e.g. K11, K12).

The SDS gels of FIG. 20 illustrate some of these proteins. Most tauconstructs have M_(r) values larger than expected from their actual mass(FIG. 20 a). A notable feature is the tendency to form dimers andoligomers. This is particularly pronounced with some constructs, forexample K12 (FIG. 20 b). The formation of dimers can already be observedby letting the protein stand for some time (FIG. 20 b, lane 2),presumably because the dimers become fixed by a disulfide bridge; thiscan be prevented by DTT (lane 1). To test this, the cross-linker PDMwhich predominantly links cysteines was used. This generates essentiallythe same products as in the absence of DTT (lane 3). Chemicalcross-linking for construct K12 (2-5 mg/ml) was carried out byincubation in 40 mM HEPES pH 7.5 with 0.5 mM DTT for 30 min at 37° C.and followed by reaction for 30 min at room temperature with 0.7 mM PDM(Sigma) or 1.5 mM MBS (Pierce) added from freshly pre-pared stocksolutions in DMSO. The reactions were quenched by addition with 5 mM DTTor 5 mM DTT and 5 mM ethanolamine, respectively. Finally, dimers andhigher oligomers can also be generated by MBS, which links cysteines andlysines (lane 4). The cross-linked species can be separated bychromatography on a Superose 12 column (FIG. 20 c), allowing the studyof a homogeneous population of dimers. For this purpose, the covalentlycross-linked dimers were separated from the monomers by gel filtrationon a Pharmacia Superose 12 FPLC column equilibrated and eluted with 50mM Tris-HCL pH 7.6 containing 0.5 M NaSCN, 0.5 M LiCl and 2 mM DTToperated at a flow rate of 0.3 ml/min. Column fractions were analysed bySDS-PAGE, pooled and concentrated by centrifugation through centricon 3microconcentrators (Amicon). The column was calibrated with the proteinsfrom the Pharmacia low molecular weight gel filtration calibration kit.Effective hydrated Stokes radii (r) of the calibration proteins weretaken from the kit's instruction manual and partition coefficients (a)were determined from the elution volumes and fitted to an equation ofthe form σ=−A log r+B, yielding the Stokes radii for the tau constructmonomers and dimers. The axial ratios were calculated following Perrin(for further details, see Cantor & Schimmel, Biophysical chemistry, PartII: Techniques for the study of biological structure and function.Freeman & Co, San Francisco, 1980) The elution profile (FIG. 20 d)yields Stokes radii of 2.5 nm for the monomer of K12, and 3.0 nm for thedimer. Given the molecular weights of 13 and 26 kDal this yields axialratios of 10 and 8, consistent with the rod-like shape observed byelectron microscopy (the equivalent lengths of prolate ellipsoids wouldbe 6.8 and 8.5 nm which underestimates the actual lengths; see below).

Other tau species show similar cross-linking results, but they aresomewhat more complex for the following reason: Tau has cysteines onlyin repeats 2 and 3 (residues Cys291 and Cys322). Repeat 2 is absent fromsome isoforms, for example htau 23 or construct K12, leaving only thelone Cys322. With Cys-Cys cross-linkers such as PDM, these molecules canonly form dimers, but no higher aggregates (FIG. 20 b, lane 3). Incontrast, bivalent molecules with two cysteines (such as htau40, K11)can form intramolecular cross-links, dimers and higher oligomers. Thisdiversity is similar to what is found after cross-linking K12 with MBS(FIG. 20 b, lane 4) because tau contains many lysines.

The conformation of several tau constructs in solution was probed byanalytical ultracentrifugation and CD spectroscopy according to standardprocedures. For example, htau40 had a sedimentation constant of 2.6S onthe mixture of tau from brain. For a globular particle of the mass ofhtau40 (45.8 kDal) one would expect ≈4.2S; the lower observed valueindicates an elongated structure with a hydrodynamic axial ratio of =15.The CD spectra of htau40 and construct K12 (FIG. 20 e) wereindistinguishable; they showed very little secondary structure. Thismeans that both the N-terminal and C-terminal domains of tau lackinternal regularity such a α-helix or β-sheet.

(b) Synthetic Paired Helical Filaments.

Tau isolated from brain tissue can self-assemble into fibrous structures(see e.g. Montejo de Garcini & Avila, J. Biochem. 102 (1987), 1415-1421;Lichtenberg-Kraag & Mandel-kow, J. Struct. Biol. 105 (1990), 46-53).This property became particularly interesting in view of the fact thattau is one of the main components of the neurofibrillary tangles ofAlzheimer's disease. In the earlier studies the relationship of thefilaments formed in vitro to the Alzheimer PHFs remained ambiguous,especially since the protein was heterogeneous. It was thereforedesirable to check if recombinant tau constructs were capable ofself-assembly. This was tested in a variety of conditions of pH, saltbuffer type, etc. Typically, solutions of tau constructs or chemicallycross-linked dimers were dialyzed against various buffers (e.g. =50-500mM MES, Tris-HCl, Tris-maleate, pH values 5-9,5-30 mM MgCl₂, CaCl₂,AlCl₃) for 12-24 hours at 4° C. The solution was briefly centrifuged(Heraeus Biofuge A, 1 min, 10,000 g) and the pellet was stored forseveral days at 4° C. and then processed for negative stain electronmicroscopy (2% uranyl acetate or 1% phosphotungstic acid). Alternativelythe solution was used for grid dialysis on gold grids following VanBruggen et al., J. Microsc. 141 (1986), 11-20. Of the constructs testedonly K11 and K12 yielded filaments resembling PHFs. The optimalconditions were 0.3-0.5 M Tris-HCl and pH 5.0-5.5, and without anyadditional salts. The results obtained with construct K12 areillustrated in FIG. 21. In the pH range of 5.0-5.5 there was extensiveformation of filaments. Their length was variable, but typically in therange of 200-1000 nm. Most appeared rather smooth, others showed aregular variation of width, with axial periodicities around 70-75 nm(arrowheads). The minimum diameter was about 8 nm and the maximum around15 nm. Short rod-like particles, about 80-150 nm in length were alsoobserved, which appeared to represent just one or two crossover periodsof the filaments (FIG. 21, middle). It was not possible to discernreliably any axial fine structure that might indicate an arrangement ofprotein subunits. This was therefore either below the resolution limitof negative stain, and/or due to lack of contrast. In general thefilaments tended to be bundled up in clusters, as if they had a highaffinity for one another (FIG. 21 a). Similar PHF-like filaments werealso obtained with K12 dimers cross-linked with PDM (FIG. 22). Thissuggests that the dimer might be an intermediate stage in filamentassembly.

Many of these features are similar to those of paired helical filamentsisolated from Alzheimer's disease brains, shown for comparison in FIG.23. Their appearance depends somewhat on the isolation procedure. FIG. 5a shows “insoluble” filaments prepared from neurofibrillary tanglesafter Wischik et al., J. Cell Biol. 100 (1985), 1905-1912. Thesefilaments are long, straight, and have a homogeneous ultrastructurecharacterized by the distinct ≈75 nm repeat. By contrast, when thefilaments are “solubilized” by sarkosyl following Greenberg & Davies,Proc. Natl. Acad. Sci. USA 83 (1990), 5827-5831, they are shorter andless homogeneous (FIG. 23 b). In particular, this preparation includesvery short particles (equivalent to about 1-2 crossover periods), andsmooth filaments that do not have the twisted appearance (reminiscent ofstraight filaments). There is a striking similarity between thesynthetic PHFs based on the repeat domain (e.g. K11, K12, K12 dimers,FIG. 21, 22) with the soluble PHFs from Alzheimer brains (FIG. 23 b),judged by three different criteria: (i) The filaments are shorter thanthe insoluble PHFs of FIG. 23 a; (ii) they are less homogeneous in theirperiodicity, and some lack the twisted appearance altogether (straightfilaments), (iii) they include very short rod-like particles, down tothe length of one crossover period.

Thus far, synthetic PHF-like fibers have only been observed withconstructs such as K12 and K11 containing essentially the repeat domain(3 or 4 repeats, FIG. 19), but not with larger tau isoforms. These dataare all consistent with the assumption that the repeat domain is thebasic unit that is capable of self-assembling into PHFs very similar tothose of Alzheimer neurofibrillary tangles. This also agrees withexperiments in several laboratories showing that the pronase-resistantcore of Alzheimer PHFs contains the repeat region (e.g. Goedert et al.,ibid., Jakes et al., EMBO J. 10 (1991), 2725-2729). It was also notedthat the filament-forming constructs were not phosphorylated so thatthis does not, in contrast to the genuine Alzheimer PHF, play a role inself-assembly here.

(c) Electron Microscopy of Tau Monomers and Dimers.

The results on the synthetic PHFs suggested that the repeat region had aspecial role in the interaction between tau molecules. It was thereforedesirable to define their structure in more detail by comparingdifferent constructs in the electron microscope. The method of choicewas metal shadowing at a very shallow angle, combined with glycerolspraying; this helps to make the particles visible which otherwise wouldnot be seen because of their low contrast.

Spraying was done following Tyler & Branton, J. Ultrastruct. Res. 71(1980), 95-102. The samples were diluted 1:10 in spraying buffer (50 mMammonium acetate pH 8.0, 150 mM NaCl, 1 mM MgCl₂, 0.1 mM EGTA), made upto 70% glycerol and sprayed onto freshly cleaved mica. The sprayedsamples were vacuum dried for 2 hours, shadowed with platinum/carbon(thickness about 1.5 nm, shadowing angle 4′) using a BAE 080T shadowingunit (Balzers Union), followed by 20-30 nm carbon. Finally the replicaswere floated off on doubly distilled water and picked up with 600 meshcopper grids.

Molecules of htau23 (352 residues, FIG. 24 a) are rod-like and have amean length of 35+7 nm (lengths summarized in Table 2 and FIG. 25). Thisvalue is less than that reported by Hirokawa et al., J. Cell Biol. 107(1988), 1449-1459, but this might be due to differences in theexperimental approach (freezing vs. glycerol spraying; a mixture of allisoforms vs. the smallest isoform). The apparent width of the metalshadowed htau23 molecules is about 3-5 nm, and the contrast is low—muchless than that of control samples (single and double stranded DNA,α-helical proteins). Careful inspection of the micrographs reveals apopulation of particles with enhanced contrast, somewhat larger diameter(5-7 nm), sometimes split into two parts, and lengths similar orslightly more than the monomer (around 40 nm). These particles areinterpreted as (nearly) juxtaposed monomers forming dimers (FIG. 24 b),consistent with the results on cross-linked dimers and antibodydecoration shown later.

Clearly longer particles are obtained with the construct T8R-1 whichaverage 58±15 nm, 23 nm more than htau23 (FIG. 24 c, 25 b). Thisconstruct contains eight repeats (a duplication of the four basic ones,FIG. 19), that is five repeats more than htau23, plus the two 29-merinserts near the N-terminus. T8R-2 has a similar length (61+17 nm), eventhough it lacks the N-terminal inserts. Construct T7R-2 also has asimilar length of 60+16 nm, even though it has only seven repeats (3+4)and no N-terminal inserts. At first sight these results appear puzzling:On the one hand, larger constructs become longer, but on the other handcertain parts of the sequence do not affect the length. Anticipating theresults below, the contradiction can be explained by a unifyinghypothesis: The length of the tau constructs is determined mainly by therepeat region; by comparison, the

N-terminal domain and the C-terminal tail are only of minor influence.The repeat region itself must be considered a unit, roughly 20-25 nmlong, whose length is approximately independent of the second repeat.The hypothesis implies that the N-terminal inserts have only a minorinfluence on the length. It predicts that constructs with 3 or 4 repeatshave roughly the same length (e.g. T7R vs. T8R), and that the additionof one repeat domain adds about 2.0-25 nm in length (as in htau23 vs.T7R or T8R).

T8R and other constructs also form particles folded into a hairpin (FIG.24 d), as if the two “units” (of four repeats each in this case) couldinteract; this is suggestive of an antiparallel arrangement, supportingthe antibody data described infra. T8R particles were also observedwhose width and contrast indicate dimers similar to htau23 (FIG. 24 e).

As in the previous cases, the repeat domain constructs that form thePHF-like fibers formed by K11 and K12 described above (FIG. 26) arerod-like. Using the criteria of thickness or contrast and the comparisonwith the dimers, K11 displays a population of low contrast monomers witha mean length of 26±5 nm (FIG. 26 a), and a population of more contrastydimers, about 32±6 nm (FIG. 26 b). This means that the two moleculesmust be juxtaposed for most of their length. For K12, monomers of length25±4 nm, (FIG. 26 d), and dimers of about 30±4 nm (FIG. 26 e) are found.The monomers have about 70-75% of the length of htau23, although theycontain only a third of the residues (FIG. 25 c, e). With bothconstructs, longer particles are found which are interpreted as dimersassociated into tetramers (FIG. 26 c,

Thus far the classification into monomers and dimers was judged byrelating the width and contrast of the particles to model structures.However, it is possible to isolate the covalently cross-linked dimers bygel chromatography and study them directly by electron microscopy andother methods. As an example, dimers of K12 cross-linked by PDM via thesingle Cys322 (FIG. 27 a) are shown. In the electron microscope, theircontrast is similar to the dimers described above; but more importantly,they are only slightly longer than the monomers (29±6 nm FIG. 27 a, FIG.25 e, g). This means that the PDM dimers are formed by two moleculeslying next to one another and nearly in register. The dimers of K12induced by MBS (34±6 nm) are also similar, except that they tend to besomewhat longer (by ≈5 nm) than those obtained with PDM, probablybecause a greater variety of Cys-Lys bonds are possible (FIG. 27 b, 25h). Taken together, the results obtained with K11 and K12 (and otherconstructs containing essentially the repeat domain) are consistent withthe hypothesis that the repeat domain forms a folding unit of ratheruniform length, independently of whether it contains 3 or 4 repeats.

For all constructs tested, the glycerol spray experiments show a certaintendency to form fibrous structures. In most cases, they are ratheruniform in diameter, they show no obvious relationship to paired helicalfilaments and may result from a distinct pathway of self-assembly.

(d) Antiparallel Alignment of Dimers

It is clear from the above data that tau and its constructs tend toalign laterally into dimers. This raised the question of polarity: Arethe particles parallel or anti-parallel? First indications came from thehairpin fold observed with the 8-repeat constructs (e.g. FIG. 24 d),suggesting antiparallel orientations of the two halves. Direct evidencefor this was obtained by labeling with the monoclonal antibody 2-4 whoseepitope is on the last repeat and therefore close to the C-terminus interms of the sequence (Dingus et al., J. Biol. Chem. 266 (1991),18854-18860). FIG. 28 a (left) shows particles of htau23 with oneantibody molecule bound. The antibodies bind at or near one end, showingthat one of the physical ends of the rod coincides roughly with theC-terminus. The lengths of the rod portions shown are similar to thoseof unlabeled htau23; in terms of apparent width, they could be monomersor dimers. In the same fields, one also finds doubly labeled particles(FIG. 28 a, right). The antibodies bind at opposite ends, proving thatthe two subunits of a dimer have opposite polarities.

The same features are found with construct K12; rodlike stubs with anantibody at one end (FIG. 28 b, left); dumb-bells, i.e. antiparalleldimers (FIG. 28 b, middle). Finally, there are particles with twoantibodies and two stubs, with a kink in the middle (pairs of“cherries,” FIG. 28 b, right). Each of the arms has roughly the lengthof a unit stub so that the particles appear equivalent to the tetramersof FIGS. 26 c and f. The interaction between the dimers at the centerappears to prevent the binding of an antibody which could otherwise beexpected there.

PDM dimers of construct K12 (formed by Cys322-CyS322 crosslinks) areshown in FIG. 28 c. Particles with one anti-body label are on the left,doubly labeled ones in the middle, showing that the chemicallycrosslinked dimer consists of antiparallel monomer. A presumptivetetramer is on the right. Essentially the same data are obtained withMBS crosslinked dimers (Cys322 to nearby Lys, FIG. 28 d).

Based on the knowledge described in this Example, in vitro methods fortesting drugs effective in dissolving Alzheimer paired helical filamentsas for testing drugs effective in the reduction or prevention of theformation of Alzheimer paired helical filaments may be developed, as isdescribed above.

EXAMPLE 10 Effect of Glycogen Synthase Kinase-3 (GSK-3) and cdk2-CyclinA on Phosphorylation of the Tau Protein

Experiments described in Examples 4 and 5 were repeated using GSK3 (alsoreferred to as phosphatase activating factor F_(A), Vandenheede et al.,J. Biol. Chem. 255 (1980), 11768-11774) as the phosphorylating enzyme.

GSK3 (α and β isoforms) were purified from bovine brain as described inVandenheede et al., ibid., with an additional Mono S chromatography stepwhich separates the two isoforms. Most experiments described here weredone with immunoprecipitates of GSK-α on TSK beads (following Van Lintet al., Analyt. Biochem. 1993, in press), but control experiments withthe β subunits showed the same behavior.

Polyclonal anti-peptide antibodies to the α and β isoforms of GSK3 wereraised in rabbits and affinity purified on peptide columns.Immunoprecipitates of GSK3 were prepared from PC-12 cytosols in 20 mMTris-HCl, 1% NP-40, 1 mM PMSF, 2 μg/ml aprotinin, 1 μg/ml leupeptin and0.2 μg/ml pepstatin. 100 μl of cytosols were incubated with 1 μl of α-or β-GSK antibodies (1 mg/ml) or control rabbit antibodies and incubatedfor 4 h at 4° C., 5 μl of TSK-protein A beads were added and incubatedfor another hour, and finally the beads were washed with 10 mg/ml BSA in20 mM Tris-HCl, 0.5 M LiCl in Tris buffer, and 20 mM Hepes pH 7.2 with10 mM MgCl₂ and 1 mM DTT. In phosphorylation assays, 2 μl of pelletswere incubated with 8 μl of substrate (3 μM) in 40 mM Hepes pH 7.2, 10mM MgCl₂, 2 mM ATP, 2 mM EGTA, 0.5 mM DTT and 1 mM PMSF.

-   (a) Time Course of Phosphorylation and Antibody Response Induced by    GSK3

FIG. 29 shows a time course of phosphorylation of htau40 with GSK3, andthe corresponding autoradiogram and immunoblots. In most respects thebehavior is similar to that obtained with the brain kinase activity orwith purified MAP kinase. Phosphorylation induces a gel shift in threemain stages; it incorporates ≈4 P_(i); it induces the reactivity ofantibodies AT8, SMI34, and SMI31, but reduces the reactivity of TAU1 andSMI33.

-   (b) Phosphorylation Sites of GSK3 on Tau

The main phosphorylation sites can be determined from anti-body epitopesand point mutants (FIG. 29). TAU1 requires that both Ser199 and Ser202are unphosphorylated, AT8 requires them both phosphorylated. Thus whenonly one of the two serines is phorphorylated these antibodies do notreact. This means that Ser199 and Ser202 both become phosphorylatedduring stage 2 (FIG. 29, panels 3,4). Similarly, antibody SMI31 requiresthe phosphorylation of both Ser396 and Ser404, which means that bothserines become phosphorylated rapidly during stage 1 (FIG. 29, panel 6).SMI33 reacts only when Ser235 is unphosphorylated so that the gradualloss of reactivity means that this residue becomes phosphorylated onlyslowly (panel 7). Together these residues would account for 5 Pi, butonly ≈4 Pi were observed by autoradiography, indicating that not all ofthese serines are phosphorylated at 100%. There are some subtledifferences in the time course of immune response, compared to MAPkinase. For example, the SMI31 reactivity sets in early and precedesthat of AT8 and SMI34, while the reactivity of SMI33 persists for alonger time, indicating that the mode of action of GSK3 is not identicalto that of MAP kinase.

Additional information can be obtained by point mutations. As shown inExamples 5 and 6, the initial strong mobility shift induced by thekinase activity from brain extracts and by MAP kinase is due to thephosphorylation of Ser454. The same is true for GSK3, as illustrated inFIG. 30 (lanes 1-3). When Ser404 is mutated into Ala, the initial rapidshift disappears, and initial phosphorylation is reduced to a low level(FIG. 30, compare lanes 2 and 5).

Another conclusion from the immunoblots is that GSK3 strongly prefersSer-Pro motifs, in contrast to MAP kinase which also affects Thr-Pro.This follows since the ≈4 Pi incorporated are needed to account for thephosphorylated epitopes. To test this construct AP11 was prepared, aderivative of htau23 where all 6 Ser-Pro are replaced with Ala-Pro (FIG.31, middle). AP11 is phosphorylated only to a minimal extent, <0.1 Piper molecule, confirming that the Thr-Pro motifs remain largelyunphosphorylated. The same result is obtained with construct AP17 (all 6Ser-Pro and 8 Thr-Pro replaced by Ala-Pro, FIG. 31, top). Anotherconstruct, K18 containing only the four repeats (FIG. 31, bottom), isalso not phosphorylated, indicating that no major sites are within themicrotubule binding region. Thus, GSK3 and MAP kinase are similar inthat they are both pro-1-line directed, but MAP kinase is also activewith respect to Thr-Pro motifs.

-   (c) GSK3 and MAP Kinase are Associated with Microtubules and with    PHFs

Considering that tau is a microtubule-associated protein one mightexpect that kinases that phosphorylate tau might be localized in thevicinity. It was therefore tested whether MAP kinase or GSK3 weremicrotube-associated proteins according to the usual criterium ofco-purification through repeated circles of assembly and disassembly.This was indeed the case. FIG. 32 b shows that both the p42 and p44isoforms of MAP kinase co-purified with porcine brain microtubules,FIGS. 32 c,d demonstrates the same for the case of GSK3 α and β.Interestingly, the microtubule-associated MAP kinase was not in anactivated state since it was not phosphorylated on Tyr (as judged byimmunoblotting, not shown).

Considering this result, it was of interest to investigate whether thekinases were also associated with Alzheimer PHFs. The immunoblots ofFIG. 33 a demonstrate that GSK3 is present in normal and in Alzheimerbrain in roughly equivalent amounts and thus resembles MAP kinase inthis respect. Moreover, the kinases co-purify directly with PHFsisolated by two different procedures, following Wischik et al., J. Cell.Biol. 100 (1985), 1905-1912 (FIG. 33 b, lane 1) and Wolozin et al.,Science 232 (1986), 648-650 (lane 2). The fact that GSK3 is associatedwith microtubules and PHFs and phosphorylates tau would suggest that thekinase might be able to affect the interaction between tau andmicrotubules. This would be in agreement with a common notion about thepathological effects of tau phosphorylation. Surprisingly, however,there was no influence on the binding. FIG. 34 shows the binding ofhtau23 to microtubules without phosphorylation, with phosphorylation byGSK3, and by the kinase activity of the brain extract. In the lattercase, there is a strong reduction in affinity, but the effect of GSK3itself is minimal.

(d) Phosphorylation of Tau by cdk2-cyclin A

The protein kinase cdk2-cyclin A (a proline-directed ser/thr kinase; seeHunter, ibid.) induces the Alzheimer-like state, as judged byphosphorylation, gel shift and antibody response. The kinase cdk2incorporated 3.5 Pi into htau40 and generated a similar shift in the gelas MAP kinase and GSK-3. The antibodies AT-8, SMI31, SMI34 recognize thephosphorylated tau, TAU-1 and SMI33 do not, again similar to MAP kinaseand GSK-3. All ser-pro motifs (Ser 199, 202, 235, 396, 405, 422) can bephosphorylated to some extent; see FIG. 46.

The preparation was as follows: Cells overproducing the cdk2/cyclin Acomplex were obtained by Dr. Piwnica Worms, Boston.

Cyclin A was fused to glutathione-S-transferase. Thus, the complex iseasily purified using glutathione agarose beads as outlined below:

Kinase Assays on Glutathione Beads:

3×10⁶ cells were infected with viruses encoding human p33^(cdk2) andhuman cyclin A (fused to glutathione-S-transferase) each at an m.o.i. of10. At 40 hours post infection, cells were rinsed (2×) in PBS. Cellswere frozen on plate at −70° C. (Cells are kept frozen until experimentsare carried out.)

Preparation of Cell Lysates:

Lyse cells in 1 ml of the following buffer:

-   -   50 mM Tris pH 7.4    -   250 mM NaCl    -   mM NaF    -   10 mM NaPPi    -   0.1% NP40    -   10% glycerol    -   protease inhibitors (0.15 units/ml aprotinin, 2 mM    -   PMSF, 20 μM leupeptin)

Plates were rocked for 15 min at 4° C., lysates were collected, placedin Eppendorf tube and spun at 10K for 10 min at 4° C. Clarified lysateswere placed in fresh Eppendorf tube.

Glutathione Precipitation

100 μl (50% slurry of agarose in PBS) of glutathione agarose (fromSigma) were added to the clarified lysate, rocked ≈1 hour at 4° C. andwere spun briefly to pellet beads.

Beads were washed two times in 1 ml of above lysis buffer and washed twotimes with incomplete kinase buffer (50 μM Tris pH 7.4, 10 mM MgCl₂). Asmuch buffer as possible was removed from the beads after the final wash.

For Kinase Assays:

Exogenous substrate was added and then complete kinase buffer was added:

-   -   50 mM Tris, pH 7.4    -   10 mM MgCl₂    -   1 mM DTT    -   10 μM unlabeled ATP    -   2 μl of gamma ³²P-labelled ATP (NEN: 3000 Ci/mM) and incubated        at 30° C. for the desired amount of time.

EXAMPLE 11 Phosphorylation of Ser 262 of Tau Protein by a Novel Kinaseand Effect Thereof on Binding to Microtubules by Tau Proteins

So far it has been shown that the Alzheimer-like state of tau proteinincludes phosphorylation of Ser-Pro and Thr-Pro motifs, and that thisstate can be mimicked by a brain extract kinase activity and by MAPkinase, as judged by the response with Alzheimer-specific antibodies. Aswill be demonstrated in the following, a crucial regulation of tau'sbinding to microtubules occurs at Ser262, a residue phosphorylated bythe brain extract activity but not by MAP kinase. A novel kinase frommammalian brain which phosphorylates this residue and thereby stronglyreduces the interaction between microtubules and tau protein hasfurthermore been purified.

Binding studies between tau and taxol-stabilized microtubules were doneas described in Example 6 This provides a direct measure of theattachment of tau to pre-formed microtubules and yields dissociationconstants and binding stoichiometries (n=tau_(bound)/tubulin dimer); thereduction in stoichiometry is the most conspicuous and reproducibleparameter. The drop in stoichiometry in a wild type tau isoform uponphosphorylation, D_(n,wt)=(n_(unphos)−n_(phos))_(wt), is taken as 100%and can be compared to the effect of phosphorylation on a mutant.D_(n,mut).

Preparation of the kinase from brain: An extract from 250 g of porcinebrain tissue was prepared and submitted to ammonium sulfateprecipitation as described in Example 2. The precipitate obtainedbetween 30 and 45% saturation was homogenized in buffer 1 (25 mMTris-HCl pH 7.4 containing 25 mM NaCl, 2 mM EGTA, 2 mM DTT, 1 mM PMSF)and dialyzed against 1 liter of this buffer with two changes overnight.Total protein concentration was determined using the Pierce BCA assaykit. After clarification of the dialysate by ultracentrifugation,portions of up to 250 mg of protein were loaded on a Mono QHR 10/10column (Pharmacia) equilibrated with buffer 1. Elution was performedwith a linear gradient of 25-500 mM NaCl in 120 ml of buffer 1 with aflow rate of 2 ml/min. Fractions were screened by phosphorylation ofbacterially expressed tau and tau constructs as described below. Activepeaks were pooled and concentrated 10 to 40-fold by centrifugationthrough Centriprep 10 microconcentrators (Amicon) and chromatographed ona Superdex 75 HiLoad 16/60 size exclusion column (Pharmacia)equilibrated and eluted with buffer 1 containing 50 mM NaCl. Activefractions were pooled and rechromatographed on a Mono Q HR 5/5 columnwith a gradient of 0-600 mM NaCl in 30 ml of buffer 1 with a flow rateof 0.5 ml/min. Active fractions were dialyzed against buffer 1 andstored at 0° C. The gel filtration column was calibrated with thePharmacia low weight marker set. Phosphorylation assays were performedas described (Steiner et al., 1990, ibid.).

In-gel assays of tau phosphorylation were done following Geahlen et al.,Anal. Biochem. 153 (1986), 151-158. MonoQ-fractions with kinase activitywere subjected to 11% SDS PAGE (0.5 mm thick slab gels). Tau protein wasadded to the separation gel solution just prior to polymerisation (finalconcentration 0.1 mg/ml). The following steps were then performed: (1)To remove SDS, the gels were washed with two changes of 20% propanol in50 mM Tris-HCl pH 8.0 for 30 min at room temperature, then 50 mMTris-HCl pH 8.0 containing 5 mM β-mercaptoethanol (=buffer A) foranother 30 min at RT. (2) The enzyme was denatured by two changes of 6 Mguanidine-HCL for 1 hour at room temperature (RT). (3) The enzyme wasrenatured by five changes of buffer A containing 0.04% Tween 40 for ≈15hours at 4° C. (4) Pre-incubation with phosphorylation buffer withoutATP for 30 min at RT (40 mM Hepes pH 7.5, 5 mM EGTA, 3 mM MgCl, 0.1 mMPMSF, 2 mM DTT). (5) Phosphorylation with added 0.1 mM ATP and 130Ci/Mol (gamma-32) ATP was performed by incubation of the gel in aplastic bag at 37° C. for 20 hours on a rotating wheel. (6) Removal ofexcess (gamma-32) ATP: The gel was washed by incubation in five changesof 300-500 ml of 5% TCA containing 1% sodiumpyrophosphate until unboundradioactivity was negligible. (7) Staining and autoradiography were doneaccording to conventional methods.

-   (a) Phosphorylation of Ser262 Strongly Reduces the Binding of Tau to    Microtubules

As shown in Example 6, when tau protein is phosphorylated by the brainextract kinase activity, the stoichiometry typically dropped from ≈0.5tau per tubulin dimer down to ≈0.1-0.15, i.e. about 3-4-fold; thiseffect on the wild type protein will be taken as 100% in this Example.The parameters affected by phosphorylation have distinct time courses. Amajor part of the gel shift occurs early (stage 1 phosphorylation, up to≈2 hours) and can be ascribed to a single site, Ser 404 (numbering ofhtau40). Most of the Alzheimer-like antibody response, as well as anadditional gel shift, sets in during stage 2 (up to ≈6 hours); a furthershift combined with more incorporation of phosphate occurs during stagethree (up to 24 hours). However, the effect on microtubule binding wasalready fully visible after stage 1. At this point, the protein boundabout two moles of P_(i) (out of a maximum of ≈5-6). About one of thesewas at Ser404, identifiable by the first gel shift. The other phosphatewas distributed among Ser202, 235, and 262, but exact quantification byautoradiograpghy and phosphopeptide sequencing was difficult.

It was therefore decided to approach the problem by site-directedmutagenesis. The Ser residues in question were replaced by Ala (makingthem non-phosphorylatable) or Asp (mimicking the negative charge of thephosphorylated state; see FIG. 35 a). These mutants were then assayedwith respect to gel shift, phosphate incorporation, and microtubulebinding (FIG. 35 b). The mutant Ser404-Ala loses its shift during stage1 phosphorylation, but the phosphorylation of this protein still has asizable effect in reducing the microtubule binding capacity (differencein stoichiometry D_(n)=0.17, i.e. 52% of the unmutated control withD_(n)=0.33). This suggests that one or more of the remaining Ser202,235, and 262 are responsible for a major fraction of the phosphorylationeffect on binding. Similar results are obtained when Ser202, 235, and396 are mutated into Ala or Asp, indicating that neither of theseresidues accounts for the low stoichiometry after phosphorylationobserved with wild type htau23. However, when Ser262 was altered, thebinding to microtubules was nearly unaffected by phosphorylation(D_(n)=0.04). In other words, it appears that mutating one residue,Ser262 in the first repeat, nearly eliminates the phosphorylationsensitivity of tau towards microtubule binding; or conversely,phosphorylation of Ser262 reduces the binding of tau to microtubulesdramatically.

-   (b) MAP Kinase Induces the Alzheimer-Like Immune Response of Tau but    does not Impair Microtubule Binding

The binding data in section (a) were obtained with a brain extract, butmost of the properties of extract phosphorylation could be induced bypurified MAP kinase from Xenopus oocytes or porcine brain. Extract andMAP kinase induce a gel shift, they have a similar time course ofphosphorylation, and both induce a similar pattern of antibody responses(including the onset of the “Alzheimer-like” response in stage 2phosphorylation). The majority of sites found with the extract are inSer-Pro motifs; all of them are phosphorylated by MAP kinase as well,plus Thr-Pro motifs, i.e. purified MAP kinase is more efficient as aPro-directed Ser/Thr kinase than the brain extract. Finally, MAP kinaseis a major phosphorylating component in the brain extracts.

However, when the effect of highly purified MAP kinase on tau'smicrotubule binding was tested it turned out to be surprisingly small(D_(n)=0.09) compared with the brain extract (D_(n)=0.31 in FIG. 36).This was consistent with the above experiments, suggesting thatphosphorylation of Ser-Pro or Thr-Pro motifs by itself was only ofsecondary importance with respect to microtubule binding.

This was tested by employing two “total” mutants, AP17 and AP18 derivedfrom htau23 (FIG. 37 a). AP18 is similar to AP17, but in addition Ser262and 356 (the two serines not followed by Pro found earlier in extractphosphorylations) were changed into Ala. While MAP kinase phosphorylatesall Ser-Pro and Thr-Pro sites of wild type htau23 (typically up to amaximum of 10-12 moles of P_(i) per htau23), AP17 incorporates at most1.4 P_(i), illustrating the high specificity of MAP kinase for Ser-Proor Thr-Pro motifs. AP17 binds tightly to microtubules, independently ofphosphorylation by MAP kinase, with similar parameters asunphosphorylated wild type htau23. The same results are obtained withAP18 and MAP kinase (<1 P_(i) incorporated).

However, when AP17 and AP18 are phosphorylated with the brain extractactivity the two mutants are dramatically different (FIG. 37 b). AP18incorporates about 0.5 P_(i) and shows only a minor reduction of thestoichiometry of tau bound to microtubules upon phosphorylation(D_(n)=0.01). AP17 incorporates ≈1.3 P_(i), and yet its reduction of thebinding of tau to microtubules upon phosphorylation is the same as thatof wild type htau23 (D_(n)=0.31).

These results made it clear that the brain extract apparently containssome phosphorylating component distinct from MAP kinase whichphosphorylates Ser262 in the first repeat of tau protein, and that thissingle Ser, when phosphorylated, is capable of dramatically altering theinteraction of tau with microtubules. By contrast, MAP kinase affectsthe other indicators of the Alzheimer state of tau, the gel shift andthe immune response.

-   (c) The 35 kDal and 70 kDal Kinases in Brain Reduces Microtubule    Binding by Phosphorylating Ser 262

The sequence around Ser 262 does not fit obvious consensus motifs ofknown kinases so that it did not seem promising to test them. Instead,the kinase was purified from the brain extract. Active fractions wereidentified by the criteria of tau phosphorylation and effect onmicrotubule binding.

The first step was ion exchange chromatography on Mono Q (FIG. 38 a),yielding 3 main peaks of kinase activity. The fractions with the largesteffect on microtubule binding were further subjected to gelchromatography (FIG. 38 b). The main active fraction eluted at an Mraround 35 kDal. This was followed by another ion exchange run. Theprotein did not bind to Mono S, suggesting an acidic pI, but it elutedas one major peak on Mono Q (FIG. 38 c). Silver stained gels of fraction9 showed a 35 kDal band with >95% purity, and minor (<5%) bands around41 kDal (FIG. 38 d, lane 5). Other fractions and an additional band at≈45 kDal, but this had no kinase activity (see below).

To determine directly which of the bands in the gel were capable ofphosphorylating tau an in-gel assay following the method of Geahlen etal., Anal. Biochem. 153 (1986), 151-158 was performed. Tau protein waspolymerized into the gel matrix, the Mono Q fractions were separated onthe gel by SDS electrophoresis, the bound proteins were renatured insitu, incubated with radioactive ATP and assayed for activity byautoradiography. FIG. 39 shows that the 35 kDal and 41 kDal bandscontained kinase activity, but not the 45 kDal band.

Quantification of the amount of phosphate incorporated into tauconstructs by the kinase yielded the following results: 3.2 P_(i) forhtau34, 3.4 for htau40, 3.3 for htau23, but only 2.8 for the mutanthtau23(Ser262→Ala). The total mutant AP17 incorporated 3.0 P_(i),indicating that Ser-Pro or Thr-Pro motifs were not targets of thekinase, and the 3-repeat construct K18 contained 1.4 P_(i).

Tau phosphorylated by the kinase is shifted upward in the SDS gel. FIG.40 a shows a comparison of different tau gel shifts and kinases. Theshift by the 35 kDal kinase is of medium magnitude (lane 2), like thatof PKA (lane 10), larger than that of CaM kinase (lane 9) but distinctlysmaller than that of MAP kinase (lane 11) which induces theAlzheimer-like immune response. The mutant Ser409-Ala (lanes 3,4) is notshifted by phosphorylation, but other mutants are (e.g. at Ser416, lanes5, 6, or at Ser404, lanes 7,8), indicating that Ser409 is the residuewhose phosphorylation by the 35 kDal kinase generates the shift. Thissame shift is found with PKA (lane 10) which also phosphorylates Ser409.Since phosphorylation sites within the repeat region generally do notproduce a shift these data confirm that the shift sites (mostly in theC-terminal tail) are distinct from the sites controlling microtubulebinding (e.g. Ser262).

The effect of the purified kinase of the binding of tau (FIG. 40 b) issimilar to that of the brain extract (FIG. 37 b). For example, thestoichiometry of htau23 is reduced by D_(n)=0.28 upon phosphorylation,but only by 0.05 in the point mutant Ser262-Ala, again emphasizing theimportance of Ser262.

A diagram of htau40, highlighting the first microtubule-binding repeatand the Ser262 that is important for microtubule-binding is depicted inFIG. 41.

A similar effect on the binding of tau to microtubules is observed whentau is phosphorylated by the 70 kDal kinase (see FIG. 45). This kinaseincorporates about 3-4 Pi into the repeat region of tau, specifically atserines 262, 293, 324, 409. It is prepared by the following steps: (a)Preparation of high spin supernatant of brain extract. (b)Chromatography on Q-Sepharose. (c) Chromatography of flowthrough onS-Sepharose. Kinase activity elutes at 250 mM NaCl. (d) Chromatographyon heparin agarose. Kinase activity elutes at 250 mM NaCl. (e) Gelfiltration. Kinase activity elutes at 70 kDal. (f) Chromatography onMono Q. Kinase activity elutes at 150 mM NaCl.

EXAMPLE 12 Dephosphorylation of Tau Protein by Phosphatases PP2a and PP1

htau 40 was phosphorylated with porcine MAP kinase (p42) and ³²P-ATPaccording to methods described throughout this specification.Subsequently, htau40 was dephosphoylated with several isoforms of PP2a(FIG. 42 A to C) as PP1 (FIG. 42D). The results show that htau40 isdephosphorylated by all isoforms of PP2a, and, although much slower, byPP1 FIG. 43 shows that upon dephosphorylation the antibody-specificepitopes disappear as well. In FIG. 44 the time course ofdephosphorylation and the Michaelis-Menten-kinetics are shown.

Thus, PP2a and PP1 serve as antagonist to MAP-kinases and may thereforebe used in pharmaceutical compositions for the treatment of Alzheimerdisease.

TABLE 1 Interactions of tau constructs with antibodies in thephosphorylated or unphosphorylated state (+ or −). The staining onimmunoblots ranges from very weak (x), to very strong, xxx. phosph.Construct +/− SM133 SM131 SM134 htau40 − xxx + xxx xxx htau23 − xxx +xxx xxx K3M − + (x) K2 − + xxx K17 − xxx + xx K10 − + xxx xxx K19 − +htau40/2235 − (x) + xxx xxx htau40/2396 − xxx + xx xxx htau40/2235/2396− (x) + xx xxx htau23/2204 − xxx + xxx xxx htau23/2396/2204 − xxx + xxxK2 − xxx + xx K5 − xxx + xx xxx K5 − xxx + xx xxx K7 − xxx + x xx K13 −xxx + xx K14 − xxx + xx K15 − xxx + xx

TABLE 2 Summary of lengths of various tau constructs. construct length(nm) s.d. (nm) number htau23 35 7 232 T8R-1 58 15 304 T8R-2 61 17 75T7R-2 60 16 73 K11 26 5 32 K12 dimer 32 6 24 K12 25 4 27 K12 dimer 30 425 K12 PDM dimer 29 6 79 K12 MBS dimer 34 6 85

1. An immunogenic composition characterized by the ability to generatean antibody which distinguishes between phosphorylated anddephosphorylated tau comprising: (a) a tau peptide consisting of the tauamino acid sequence Lys-Ile-Gly-Ser-Thr-Glu-Asn-Leu-Lys (residues259-267 in SEQ ID NO: 1) conjugated to (b) a carrier molecule, whereinthe carrier molecule induces or enhances an immune response to thepeptide of (a).
 2. The immunogenic composition of claim 1, wherein theSer (residue 262 of SEQ ID NO:1) is phosphorylated.
 3. A method ofproducing an antibody which distinguishes between phosphorylated anddephosphorylated tau, the method comprising administering to an animalan antibody-producing amount of an immunogenic composition comprising:(a) a peptide consisting of the tau amino acid sequenceLys-Ile-Gly-Ser-Thr-Glu-Asn-Leu-Lys (residues 259-267 in SEQ ID NO: I)conjugated to (b) a carrier molecule, wherein the carrier moleculeinduces or enhances an immune response to the peptide of (a).
 4. Amethod of producing an antibody which distinguishes betweenphosphorylated and dephosphorylated tau, the method comprisingadministering to an animal an antibody-producing amount of animmunogenic composition comprising: (a) a peptide consisting of the tauamino acid sequence Lys-Ile-Gly-Ser-Thr-Glu-Asn-Leu-Lys (residues259-267 in SEQ ID NO: 1) conjugated to (b) a carrier molecule, whereinthe carrier molecule induces or enhances an immune response to thepeptide of (a); wherein the Ser (residue 262 of SEQ ID NO: 1) isphosphorylated.